Methods in Enzymology Vol 356: Laser Capture in Microscopy and Microdissection

Methods in Enzymology Volume 356 LASER CAPTURE MICROSCOPY AND MICRODISSECTION METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF ...

Author: P. Michael Conn (Editor)

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Methods in Enzymology Volume 356 LASER CAPTURE MICROSCOPY AND MICRODISSECTION

METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF

John N. Abelson

Melvin I. Simon

DIVISION OF BIOLOGY CALIFORNIA INSTITUTE OF TECHNOLOGY PASADENA, CALIFORNIA

FOUNDING EDITORS

Sidney P. Colowick and Nathan O. Kaplan

Methods in Enzymology Volume 356

Laser Capture Microscopy and Microdissection EDITED BY

P. Michael Conn OREGON HEALTH AND SCIENCE UNIVERSITY PORTLAND, OREGON, AND OREGON NATIONAL PRIMATE RESEARCH CENTER BEAVERTON, OREGON

Amsterdam Boston London New York Oxford Paris San Diego San Francisco Singapore Sydney Tokyo

∞ This book is printed on acid-free paper. C 2002, Elsevier Science (USA). Copyright

All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2002 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0076-6879/2002 $35.00 Explicit permission from Academic Press is not required to reproduce a maximum of two figures or tables from an Academic Press chapter in another scientific or research publication provided that the material has not been credited to another source and that full credit to the Academic Press chapter is given.

Academic Press An imprint of Elsevier Science. 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.academicpress.com

Academic Press 84 Theobalds Road, London WC1X 8RR, UK http://www.academicpress.com International Standard Book Number: 0-12-182259-1 PRINTED IN THE UNITED STATES OF AMERICA 02 03 04 05 06 07 SB 9 8 7 6 5 4

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Table of Contents

CONTRIBUTORS TO VOLUME 356 . . . . . . . . . . . . . . . .

ix

PREFACE . . . . . . . . . . . . . . . . . . . . . . .

xiii

. . . . . . . . . . . . . . . . . . .

xv

VOLUMES IN SERIES

Section I. Basic Principles 1. Comparison of Current Equipment

ANDA CORNEA AND ALISON MUNGENAST

2. Laser Capture Microdissection and Its Applica- JAMES L. WITTLIFF AND tions in Genomics and Proteomics MARK G. ERLANDER 3. Going in Vivo with Laser Microdissection

ANETTE MAYER, MONIKA STICH, DIETER BROCKSCH, ¨ KARIN SCHUTZE , AND GEORGIA LAHR

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25

4. Use of Laser Capture Microdissection to Selec- RACHEL A. CRAVEN AND tively Obtain Distinct Populations of Cells for ROSAMONDE E. BANKS Proteomic Analysis

33

5. Optimized Tissue Processing and Staining for LORA E. HUANG, Laser Capture Microdissection and Nucleic VERONICA LUZZI, Acid Retrieval TORSTEN EHRIG, VICTORIA HOLTSCHLAG, AND MARK A. WATSON

49

6. Fluorescence in Situ Hybridization of LCM- DOUGLAS J. DEMETRICK, Isolated Nuclei from Paraffin Sections SABITA K. MURTHY, AND LISA M. DIFRANCESCO

63

7. Immunoblotting of Single Cell Types Isolated LIVIA CASCIOLA-ROSEN AND from Frozen Sections by Laser Microdissection KANNEBOYINA NAGARAJU

70

8. Noncontact Laser Catapulting: A Basic Procedure GABRIELA WESTPHAL, for Functional Genomics and Proteomics RENATE BURGEMEISTER, GABRIELE FRIEDEMANN, AXEL WELLMANN, NICOLAS WERNERT, VOLKER WOLLSCHEID, BERND BECKER, THOMAS VOGT, ¨ RUTH KNUCHEL , WILHELM STOLZ, ¨ AND KARIN SCHUTZE 80 v

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TABLE OF CONTENTS

9. Internal Standards for Laser Microdissection

LUDGER FINK AND RAINER MARIA BOHLE

10. Methacarn: A Fixation Tool for Multipurpose MAKOTO SHIBUTANI AND Genetic Analysis from Paraffin-Embedded CHIKAKO UNEYAMA Tissues

99

114

Section II. Specialized Uses 11. Use of Laser Capture Microdissection for Clonal VALE´ RIE PARADIS Analysis AND PIERRE BEDOSSA

129

12. Laser Capture Microdissection in Carcinoma YEN-LI LO AND Analysis CHEN-YANG SHEN

137

´ 13. Laser Capture Microdissection to Assess Devel- CARLOS A. SUAREZ -QUIAN, opment OSCAR M. TIRADO, FRANCINA MUNELL, ´ AND JAUME REVENTOS

145

14. Application of Laser Capture Microdissection to K. K. JAIN Proteomics

157

15. Laser Capture Microdissection of Mouse Intestine: Characterizing mRNA and Protein Expression, and Profiling Intermediary Metabolism in Specified Cell Populations

THADDEUS S. STAPPENBECK, LORA V. HOOPER, JILL K. MANCHESTER, MELISSA H. WONG, AND JEFFREY I. GORDON 168

16. Laser Capture Microdissection in Pathology

FALKO FEND, KATJA SPECHT, MARCUS KREMER, AND LETICIA QUINTANILLA-MARTÍNEZ 197

17. Use of Laser Capture Microscopy in the Analysis MERAL J. ARIN AND of Mouse Models of Human Diseases DENNIS R. ROOP

208

18. Use of Laser Microdissection in Complex Tissue HOLGER S. WILLENBERG, RHODRI WALTERS, AND STEFAN R. BORNSTEIN

217

19. Assessment of Clonal Relationships in Malignant KOJO S. J. Lymphomas ELENITOBA-JOHNSON

225

20. Comparison of Normal and Tumor Cells by Laser JAUME MORA, Capture Microdissection MUZAFFAR AKRAM, AND WILLIAM L. GERALD

241

21. Analysis of Folliculostellate Cells by Laser RICARDO V. LLOYD, Capture Microdissection and Reverse Trans- LONG JIN, cription–Polymerase Chain Reaction (LCM- KATHARINA H. RUEBEL, RT/PCR) AND JILL M. BAYLISS

249

TABLE OF CONTENTS

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Section III. Genetic Applications 22. Analysis of Gene Expression 23. Analysis of Specific Gene Expression

JANETTE K. BURGESS AND BRENT E. MCPARLAND

259

GEORGIA LAHR, ANNA STARZINSKI-POWITZ, AND ANETTE MAYER

271

24. Gene Discovery with Laser Capture Microscopy MAURICIO NEIRA AND EDWIN AZEN

282

25. DNA Fingerprinting from Cells Captured by Laser YONGYUT SIRIVATANAUKSORN, Microdissection VORAPAN SIRIVATANAUKSORN, AND NICHOLAS R. LEMOINE 289 26. Single Cell PCR in Laser Capture Microscopy

SINUHE HAHN, XIAO YAN ZHONG, AND WOLFGANG HOLZGREVE

295

27. Assessment of Genetic Heterogeneity in Tumors DAVE S. B. HOON, Using Laser Capture Microdissection AKIHIDE FUJIMOTO, SHERRY SHU, AND BRET TABACK

302

28. Gene Mutations: Analysis in Proliferative Pro- HITOSHI TAKAYAMA, static Diseases Using Laser Capture Microdis- NORIO NONOMURA, AND section KATSUYUKI AOZASA

309

29. Use of Laser Capture Microdissection-Generated HIROE OHYAMA, Targets for Hybridization of High-Density Oli- MAMATHA MAHADEVAPPA, gonucleotide Arrays HEIKKI LUUKKAA, RANDY TODD, JANET A. WARRINGTON, AND DAVID T. W. WONG

323

˚ SA PERSSON, 30. Single Cell Gene Mutation Analysis Using Laser- A ¨ Assisted Microdissection of Tissue Sections HELENA BACKVALL , FREDRIK PONTE´ N, MATHIAS UHLE´ N, AND JOAKIM LUNDEBERG

334

31. Methylation in Gene Promoters: Assessment after ARTHUR R. BROTHMAN Laser Capture Microdissection AND JIANG CUI

343

AUTHOR INDEX . . . . . . . . . . . . . . . . . . . . .

353

SUBJECT INDEX . . . . . . . . . . . . . . . . . . . . .

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This Page Intentionally Left Blank

Contributors to Volume 356 Article numbers are in parentheses following the names of contributors. Affiliations listed are current.

ARTHUR R. BROTHMAN (31), Departments of Pediatrics and Human Genetics, University of Utah Health Sciences Center, Salt Lake City, Utah 84132

MUZAFFAR AKRAM (20), Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 KATSUYUKI AOZASA (28), Department of Pathology, Osaka University Medical School, Suita, Osaka 565-0871, Japan MERAL J. ARIN (17), Department of Dermatology, University of Cologne, 50924 Cologne, Germany EDWIN AZEN (24), Department of Medicine, University of Wisconsin, Madison, Wisconsin 53706 ¨ (30), Department of HELENA BACKVALL Genetics and Pathology, University Hospital, S-751 85 Uppsala, Sweden ROSAMONDE E. BANKS (4), Cancer Research UK Clinical Centre, St. James University Hospital, Leeds LS9 7TF, United Kingdom JILL M. BAYLISS (21), Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 55905 BERND BECKER (8), Department of Dermatology, Institute of Pathology, University of Regensburg, 93053 Regensburg, Germany PIERRE BEDOSSA (11), Faculty of Pharmacy, CNRS ESA 8067, 75005 Paris, France RAINER MARIA BOHLE (9), Institute of Pathology, Justus-Liebig-Universität Giessen, 35392 Giessen, Germany STEFAN R. BORNSTEIN (18), Department of Endocrinology, University of Düsseldorf, D-40225 Düsseldorf, Germany DIETER BROCKSCH (3), Servicebereich Corporate Communications, Carl Zeiss, D73447 Oberkochen, Germany

RENATE BURGEMEISTER (8), P.A.L.M. Microlaser Technologies AG, 82347 Bernried, Germany JANETTE K. BURGESS (22), Respiratory Research Group, Department of Pharmacology, University of Sydney, Sydney, New South Wales, 2006 Australia LIVIA CASCIOLA-ROSEN (7), Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 ANDA CORNEA (1), Oregon National Primate Research Center, Beaverton, Oregon 97006 RACHEL A. CRAVEN (4), Cancer Research UK Clinical Centre, St. James University Hospital, Leeds LS9 7TF, United Kingdom JIANG CUI (31), Departments of Pediatrics and Human Genetics, University of Utah Health Sciences Center, Salt Lake City, Utah 84132 DOUGLAS J. DEMETRICK (6), Calgary Laboratory Services, Departments of Pathology, Oncology, and Medical Biochemistry, University of Calgary, Calgary, Alberta T2N 1N4, Canada LISA M. DIFRANCESCO (6), Calgary Laboratory Services, Departments of Pathology, Oncology, and Medical Biochemistry, University of Calgary, Calgary, Alberta T2N 1N4, Canada ix

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CONTRIBUTORS TO VOLUME

TORSTEN EHRIG (5), Department of Dermatology, University of Texas Southwestern Medical Center, Dallas, Texas 75235 KOJO S. J. ELENITOBA-JOHNSON (19), Division of Anatomic Pathology, University of Utah Health Sciences Center, Salt Lake City, Utah 84132 MARK G. ERLANDER (2), Arcturus Applied Genomics, West Carlsbad, California 92008 FALKO FEND (16), Institute of Pathology, Technical University Munich, D-81675 Munich, Germany

356

LORA V. HOOPER (15), Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110 LORA E. HUANG (5), Department of Internal Medicine, University of Iowa, Iowa City, Iowa 52246 K. K. JAIN (14), Jain PharmaBiotech, CH4057 Basel, Switzerland LONG JIN (21), Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 55905

LUDGER FINK (9), Institute of Pathology, Justus-Liebig-Universität Giessen, 35392 Giessen, Germany

¨ RUTH KNUCHEL (8), Department of Dermatology, Institute of Pathology, University of Regensburg, 93053 Regensburg, Germany

GABRIELE FRIEDEMANN (8), P.A.L.M. Microlaser Technologies AG, 82347 Bernried, Germany

MARCUS KREMER (16), Institute of Pathology, Technical University Munich, D-81675 Munich, Germany

AKIHIDE FUJIMOTO (27), Department of Molecular Oncology, John Wayne Cancer Institute, Santa Monica, California 90404

GEORGIA LAHR (3, 23), Laser Laboratory and Department of Molecular Biology, Staedtisches Krankenhaus MünchenHarlaching, D-81545 Munich, Germany

WILLIAM L. GERALD (20), Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 JEFFREY I. GORDON (15), Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110 SINUHE HAHN (26), Laboratory for Prenatal Medicine, Department of Obstetrics and Gynecology, University of Basel, CH-4031 Basel, Switzerland VICTORIA HOLTSCHLAG (5), The Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine, St. Louis, Missouri 63110 WOLFGANG HOLZGREVE (26), Laboratory for Prenatal Medicine, Department of Obstetrics and Gynecology, University of Basel, CH-4031 Basel, Switzerland DAVE S. B. HOON (27), Department of Molecular Oncology, John Wayne Cancer Institute, Santa Monica, California 90404

NICHOLAS R. LEMOINE (25), Cancer Research UK Molecular Oncology Unit, Department of Cancer Medicine, Imperial College of Science, Technology, and Medicine, London W12 ONN, United Kingdom RICARDO V. LLOYD (21), Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 55905 YEN-LI LO (12), Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan JOAKIM LUNDEBERG (30), Department of Biotechnology, Royal Institute of Technology (KTH), SCFAB, S-106 91 Stockholm, Sweden HEIKKI LUUKKAA (29), Division of Oral Pathology, Department of Oral Medicine and Diagnostic Sciences, Harvard School of Dental Medicine, Boston, Massachusetts 02115


VERONICA LUZZI (5), Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110 MAMATHA MAHADEVAPPA (29), Affymetrix Inc., Santa Clara, California 95051 JILL K. MANCHESTER (15), Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110 ANETTE MAYER (3, 23), Humangenetik für Biologen, Universität Frankfurt, D60054 Frankfurt/Main, Germany BRENT E. MCPARLAND (22), Department of Pathology, University of Sydney, Sydney, New South Wales, 2006 Australia JAUME MORA (20), Department of Hematology and Oncology, Hospital Sant Joan de Deu de Barcelona, Barcelona, Spain FRANCINA MUNELL (13), Unitat de Recerca Biomèdica, Hospital MaternoInfantil, Vall d’Hebron Hospital, 08035 Barcelona, Spain ALISON MUNGENAST (1), Oregon Health and Science University, Portland, Oregon 97201, and Oregon National Primate Research Center, Beaverton, Oregon 97006 SABITA K. MURTHY (6), Departments of Pathology, Oncology, and Medical Biochemistry, University of Calgary, Calgary, Alberta T2N 1N4, Canada KANNEBOYINA NAGARAJU (7), Division of Rheumatology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 MAURICIO NEIRA (24), Department of Zoology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada NORIO NONOMURA (28), Department of Urology, Osaka University Medical School, Suita, Osaka 565-0871, Japan HIROE OHYAMA (29), Division of Oral Pathology, Department of Oral Medicine and Diagnostic Sciences, Harvard School of Dental Medicine, Boston, Massachusetts 02115

356

xi

VALE´ RIE PARADIS (11), Department of Pathology, Beaujon Hospital, 92110 Clichy, France ˚ SA PERSSON (30), Department of BiotechA nology, Royal Institute of Technology (KTH), SCFAB, S-106 91 Stockholm, Sweden FREDRIK PONTE´ N (30), Department of Genetics and Pathology, University Hospital, S-751 85 Uppsala, Sweden LETICIA QUINTANILLA-MARTÍNEZ (16), Department of Pathology, GSF Research Center for Environment and Health, D-85758 Oberschleissheim, Germany ´ (13), Unitat de ReJAUME REVENTOS cerca Biomèdica, Hospital MaternoInfantil, Vall d’Hebron Hospital, 08035 Barcelona, Spain DENNIS R. ROOP (17), Departments of Molecular and Cellular Biology and Dermatology, Baylor College of Medicine, Houston, Texas 77030 KATHARINA H. RUEBEL (21), Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 55905 ¨ KARIN SCHUTZE (3, 8), P.A.L.M. Microlaser Technologies AG, 82347 Bernried, Germany CHEN-YANG SHEN (12), Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan MAKOTO SHIBUTANI (10), Division of Pathology, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan SHERRY SHU (27), Department of Molecular Oncology, John Wayne Cancer Institute, Santa Monica, California 90404 VORAPAN SIRIVATANAUKSORN (25), Faculty of Medicine, Mahidol University, Bangkok 10700, Thailand YONGYUT SIRIVATANAUKSORN (25), Faculty of Medicine, Mahidol University, Bangkok 10700, Thailand

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KATJA SPECHT (16), Institute of Pathology, Technical University Munich, D-81675 Munich, Germany THADDEUS S. STAPPENBECK (15), Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110 ANNA STARZINSKI-POWITZ (23), Humangenetik für Biologen, Universität Frankfurt, D-60054 Frankfurt/Main, Germany MONIKA STICH (3), Laser Laboratory and Department of Molecular Biology, Staedtisches Krankenhaus MünchenHarlaching, D-81545 Munich, Germany WILHELM STOLZ (8), Department of Dermatology, Institute of Pathology, University of Regensburg, 93053 Regensburg, Germany ´ CARLOS A. SUAREZ -QUIAN (13), Department of Cell Biology, Georgetown University Medical School, Washington, D.C. 20007 BRET TABACK (27), Department of Molecular Oncology, John Wayne Cancer Institute, Santa Monica, California 90404 HITOSHI TAKAYAMA (28), Department of Pathology, Osaka University Medical School, Suita, Osaka 565-0871, Japan OSCAR M. TIRADO (13), Unitat de Recerca Biomèdica, Hospital MaternoInfantil, Vall d’Hebron Hospital, 08035 Barcelona, Spain RANDY TODD (29), Division of Oral Pathology, Department of Oral Medicine and Diagnostic Sciences, Harvard School of Dental Medicine, Boston, Massachusetts 02115 MATHIAS UHLE´ N (30), Department of Biotechnology, Royal Institute of Technology (KTH), SCAFB, S-106 91 Stockholm, Sweden CHIKAKO UNEYAMA (10), Division of Pathology, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan

356

THOMAS VOGT (8), Department of Dermatology, Institute of Pathology, University of Regensburg, 93053 Regensburg, Germany RHODRI WALTERS (18), Department of Endocrinology, University of Düsseldorf, D40225 Düsseldorf, Germany JANET A. WARRINGTON (29), Affymetrix Inc., Santa Clara, California 95051 MARK A. WATSON (5), Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110 AXEL WELLMANN (8), Institute of Pathology, University of Bonn, 53011 Bonn, Germany NICOLAS WERNERT (8), Institute of Pathology, University of Bonn, 53011 Bonn, Germany GABRIELA WESTPHAL (8), P.A.L.M. Microlaser Technologies AG, 82347 Bernried, Germany HOLGER S. WILLENBERG (18), Department of Endocrinology, University of Düsseldorf, D-40225 Düsseldorf, Germany JAMES L. WITTLIFF (2), Hormone Receptor Laboratory, James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky 40202 VOLKER WOLLSCHEID (8), Ciphergen Biosystems Ltd., Surrey Technology Centre, Guildford, Surrey GU2 7YG, United Kingdom DAVID T. W. WONG (29), University of California School of Dentistry, Dental Research Institute, Los Angeles, California 90095 MELISSA H. WONG (15), Department of Dermatology, Cell and Development Biology, Oregon Health and Science University, Portland, Oregon 97201 XIAO YAN ZHONG (26), Laboratory for Prenatal Medicine, Department of Obstetrics and Gynecology, University of Basel, CH-4031 Basel, Switzerland

Preface

Five years ago few people had heard of “laser microdissection” or “laser capture microscopy.” Now most major institutions have it as a core facility. This volume documents many diverse uses for this technique in disciplines that broadly span biology. The methods presented include shortcuts and conveniences not included in the sources from which they were taken. To the degree possible, we have included information needed to select equipment, prepare samples, and analyze data. The techniques are described in a context that allows comparisons to other related methodologies. The authors were encouraged to do this in the belief that such comparisons are valuable to readers who must adapt extant procedures to new systems. Also, so far as possible, methodologies are presented in a manner that stresses their general applicability and potential limitations. Although for various reasons some topics are not covered, the volume provides a substantial and current overview of the extant methodology in the field and a view of its rapid development. Particular thanks go to the authors for their attention to meeting deadlines and for maintaining high standards of quality, to the series editors for their encouragement, and to the staff of Academic Press for their help and timely publication of this volume. P. MICHAEL CONN

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METHODS IN ENZYMOLOGY VOLUME I. Preparation and Assay of Enzymes Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME II. Preparation and Assay of Enzymes Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME III. Preparation and Assay of Substrates Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME IV. Special Techniques for the Enzymologist Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME V. Preparation and Assay of Enzymes Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME VI. Preparation and Assay of Enzymes (Continued ) Preparation and Assay of Substrates Special Techniques Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME VII. Cumulative Subject Index Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME VIII. Complex Carbohydrates Edited by ELIZABETH F. NEUFELD AND VICTOR GINSBURG VOLUME IX. Carbohydrate Metabolism Edited by WILLIS A. WOOD VOLUME X. Oxidation and Phosphorylation Edited by RONALD W. ESTABROOK AND MAYNARD E. PULLMAN VOLUME XI. Enzyme Structure Edited by C. H. W. HIRS VOLUME XII. Nucleic Acids (Parts A and B) Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XIII. Citric Acid Cycle Edited by J. M. LOWENSTEIN VOLUME XIV. Lipids Edited by J. M. LOWENSTEIN VOLUME XV. Steroids and Terpenoids Edited by RAYMOND B. CLAYTON VOLUME XVI. Fast Reactions Edited by KENNETH KUSTIN xv

xvi

METHODS IN ENZYMOLOZY

VOLUME XVII. Metabolism of Amino Acids and Amines (Parts A and B) Edited by HERBERT TABOR AND CELIA WHITE TABOR VOLUME XVIII. Vitamins and Coenzymes (Parts A, B, and C) Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME XIX. Proteolytic Enzymes Edited by GERTRUDE E. PERLMANN AND LASZLO LORAND VOLUME XX. Nucleic Acids and Protein Synthesis (Part C) Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME XXI. Nucleic Acids (Part D) Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XXII. Enzyme Purification and Related Techniques Edited by WILLIAM B. JAKOBY VOLUME XXIII. Photosynthesis (Part A) Edited by ANTHONY SAN PIETRO VOLUME XXIV. Photosynthesis and Nitrogen Fixation (Part B) Edited by ANTHONY SAN PIETRO VOLUME XXV. Enzyme Structure (Part B) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVI. Enzyme Structure (Part C) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVII. Enzyme Structure (Part D) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVIII. Complex Carbohydrates (Part B) Edited by VICTOR GINSBURG VOLUME XXIX. Nucleic Acids and Protein Synthesis (Part E) Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XXX. Nucleic Acids and Protein Synthesis (Part F) Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME XXXI. Biomembranes (Part A) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME XXXII. Biomembranes (Part B) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME XXXIII. Cumulative Subject Index Volumes I-XXX Edited by MARTHA G. DENNIS AND EDWARD A. DENNIS VOLUME XXXIV. Affinity Techniques (Enzyme Purification: Part B) Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK VOLUME XXXV. Lipids (Part B) Edited by JOHN M. LOWENSTEIN


xvii

VOLUME XXXVI. Hormone Action (Part A: Steroid Hormones) Edited by BERT W. O’MALLEY AND JOEL G. HARDMAN VOLUME XXXVII. Hormone Action (Part B: Peptide Hormones) Edited by BERT W. O’MALLEY AND JOEL G. HARDMAN VOLUME XXXVIII. Hormone Action (Part C: Cyclic Nucleotides) Edited by JOEL G. HARDMAN AND BERT W. O’MALLEY VOLUME XXXIX. Hormone Action (Part D: Isolated Cells, Tissues, and Organ Systems) Edited by JOEL G. HARDMAN AND BERT W. O’MALLEY VOLUME XL. Hormone Action (Part E: Nuclear Structure and Function) Edited by BERT W. O’MALLEY AND JOEL G. HARDMAN VOLUME XLI. Carbohydrate Metabolism (Part B) Edited by W. A. WOOD VOLUME XLII. Carbohydrate Metabolism (Part C) Edited by W. A. WOOD VOLUME XLIII. Antibiotics Edited by JOHN H. HASH VOLUME XLIV. Immobilized Enzymes Edited by KLAUS MOSBACH VOLUME XLV. Proteolytic Enzymes (Part B) Edited by LASZLO LORAND VOLUME XLVI. Affinity Labeling Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK VOLUME XLVII. Enzyme Structure (Part E) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XLVIII. Enzyme Structure (Part F) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XLIX. Enzyme Structure (Part G) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME L. Complex Carbohydrates (Part C) Edited by VICTOR GINSBURG VOLUME LI. Purine and Pyrimidine Nucleotide Metabolism Edited by PATRICIA A. HOFFEE AND MARY ELLEN JONES VOLUME LII. Biomembranes (Part C: Biological Oxidations) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LIII. Biomembranes (Part D: Biological Oxidations) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LIV. Biomembranes (Part E: Biological Oxidations) Edited by SIDNEY FLEISCHER AND LESTER PACKER

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VOLUME LV. Biomembranes (Part F: Bioenergetics) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LVI. Biomembranes (Part G: Bioenergetics) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LVII. Bioluminescence and Chemiluminescence Edited by MARLENE A. DELUCA VOLUME LVIII. Cell Culture Edited by WILLIAM B. JAKOBY AND IRA PASTAN VOLUME LIX. Nucleic Acids and Protein Synthesis (Part G) Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME LX. Nucleic Acids and Protein Synthesis (Part H) Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME 61. Enzyme Structure (Part H) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 62. Vitamins and Coenzymes (Part D) Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME 63. Enzyme Kinetics and Mechanism (Part A: Initial Rate and Inhibitor Methods) Edited by DANIEL L. PURICH VOLUME 64. Enzyme Kinetics and Mechanism (Part B: Isotopic Probes and Complex Enzyme Systems) Edited by DANIEL L. PURICH VOLUME 65. Nucleic Acids (Part I) Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME 66. Vitamins and Coenzymes (Part E) Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME 67. Vitamins and Coenzymes (Part F) Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME 68. Recombinant DNA Edited by RAY WU VOLUME 69. Photosynthesis and Nitrogen Fixation (Part C) Edited by ANTHONY SAN PIETRO VOLUME 70. Immunochemical Techniques (Part A) Edited by HELEN VAN VUNAKIS AND JOHN J. LANGONE VOLUME 71. Lipids (Part C) Edited by JOHN M. LOWENSTEIN VOLUME 72. Lipids (Part D) Edited by JOHN M. LOWENSTEIN


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VOLUME 73. Immunochemical Techniques (Part B) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 74. Immunochemical Techniques (Part C) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 75. Cumulative Subject Index Volumes XXXI, XXXII, XXXIV–LX Edited by EDWARD A. DENNIS AND MARTHA G. DENNIS VOLUME 76. Hemoglobins Edited by ERALDO ANTONINI, LUIGI ROSSI-BERNARDI, AND EMILIA CHIANCONE VOLUME 77. Detoxication and Drug Metabolism Edited by WILLIAM B. JAKOBY VOLUME 78. Interferons (Part A) Edited by SIDNEY PESTKA VOLUME 79. Interferons (Part B) Edited by SIDNEY PESTKA VOLUME 80. Proteolytic Enzymes (Part C) Edited by LASZLO LORAND VOLUME 81. Biomembranes (Part H: Visual Pigments and Purple Membranes, I) Edited by LESTER PACKER VOLUME 82. Structural and Contractile Proteins (Part A: Extracellular Matrix) Edited by LEON W. CUNNINGHAM AND DIXIE W. FREDERIKSEN VOLUME 83. Complex Carbohydrates (Part D) Edited by VICTOR GINSBURG VOLUME 84. Immunochemical Techniques (Part D: Selected Immunoassays) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 85. Structural and Contractile Proteins (Part B: The Contractile Apparatus and the Cytoskeleton) Edited by DIXIE W. FREDERIKSEN AND LEON W. CUNNINGHAM VOLUME 86. Prostaglandins and Arachidonate Metabolites Edited by WILLIAM E. M. LANDS AND WILLIAM L. SMITH VOLUME 87. Enzyme Kinetics and Mechanism (Part C: Intermediates, Stereochemistry, and Rate Studies) Edited by DANIEL L. PURICH VOLUME 88. Biomembranes (Part I: Visual Pigments and Purple Membranes, II) Edited by LESTER PACKER VOLUME 89. Carbohydrate Metabolism (Part D) Edited by WILLIS A. WOOD VOLUME 90. Carbohydrate Metabolism (Part E) Edited by WILLIS A. WOOD

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VOLUME 91. Enzyme Structure (Part I) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 92. Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 93. Immunochemical Techniques (Part F: Conventional Antibodies, Fc Receptors, and Cytotoxicity) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 94. Polyamines Edited by HERBERT TABOR AND CELIA WHITE TABOR VOLUME 95. Cumulative Subject Index Volumes 61–74, 76–80 Edited by EDWARD A. DENNIS AND MARTHA G. DENNIS VOLUME 96. Biomembranes [Part J: Membrane Biogenesis: Assembly and Targeting (General Methods; Eukaryotes)] Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 97. Biomembranes [Part K: Membrane Biogenesis: Assembly and Targeting (Prokaryotes, Mitochondria, and Chloroplasts)] Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 98. Biomembranes (Part L: Membrane Biogenesis: Processing and Recycling) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 99. Hormone Action (Part F: Protein Kinases) Edited by JACKIE D. CORBIN AND JOEL G. HARDMAN VOLUME 100. Recombinant DNA (Part B) Edited by RAY WU, LAWRENCE GROSSMAN, AND KIVIE MOLDAVE VOLUME 101. Recombinant DNA (Part C) Edited by RAY WU, LAWRENCE GROSSMAN, AND KIVIE MOLDAVE VOLUME 102. Hormone Action (Part G: Calmodulin and Calcium-Binding Proteins) Edited by ANTHONY R. MEANS AND BERT W. O’MALLEY VOLUME 103. Hormone Action (Part H: Neuroendocrine Peptides) Edited by P. MICHAEL CONN VOLUME 104. Enzyme Purification and Related Techniques (Part C) Edited by WILLIAM B. JAKOBY VOLUME 105. Oxygen Radicals in Biological Systems Edited by LESTER PACKER VOLUME 106. Posttranslational Modifications (Part A) Edited by FINN WOLD AND KIVIE MOLDAVE VOLUME 107. Posttranslational Modifications (Part B) Edited by FINN WOLD AND KIVIE MOLDAVE


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VOLUME 108. Immunochemical Techniques (Part G: Separation and Characterization of Lymphoid Cells) Edited by GIOVANNI DI SABATO, JOHN J. LANGONE, AND HELEN VAN VUNAKIS VOLUME 109. Hormone Action (Part I: Peptide Hormones) Edited by LUTZ BIRNBAUMER AND BERT W. O’MALLEY VOLUME 110. Steroids and Isoprenoids (Part A) Edited by JOHN H. LAW AND HANS C. RILLING VOLUME 111. Steroids and Isoprenoids (Part B) Edited by JOHN H. LAW AND HANS C. RILLING VOLUME 112. Drug and Enzyme Targeting (Part A) Edited by KENNETH J. WIDDER AND RALPH GREEN VOLUME 113. Glutamate, Glutamine, Glutathione, and Related Compounds Edited by ALTON MEISTER VOLUME 114. Diffraction Methods for Biological Macromolecules (Part A) Edited by HAROLD W. WYCKOFF, C. H. W. HIRS, AND SERGE N. TIMASHEFF VOLUME 115. Diffraction Methods for Biological Macromolecules (Part B) Edited by HAROLD W. WYCKOFF, C. H. W. HIRS, AND SERGE N. TIMASHEFF VOLUME 116. Immunochemical Techniques (Part H: Effectors and Mediators of Lymphoid Cell Functions) Edited by GIOVANNI DI SABATO, JOHN J. LANGONE, AND HELEN VAN VUNAKIS VOLUME 117. Enzyme Structure (Part J) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 118. Plant Molecular Biology Edited by ARTHUR WEISSBACH AND HERBERT WEISSBACH VOLUME 119. Interferons (Part C) Edited by SIDNEY PESTKA VOLUME 120. Cumulative Subject Index Volumes 81–94, 96–101 VOLUME 121. Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 122. Vitamins and Coenzymes (Part G) Edited by FRANK CHYTIL AND DONALD B. MCCORMICK VOLUME 123. Vitamins and Coenzymes (Part H) Edited by FRANK CHYTIL AND DONALD B. MCCORMICK VOLUME 124. Hormone Action (Part J: Neuroendocrine Peptides) Edited by P. MICHAEL CONN VOLUME 125. Biomembranes (Part M: Transport in Bacteria, Mitochondria, and Chloroplasts: General Approaches and Transport Systems) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER

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VOLUME 126. Biomembranes (Part N: Transport in Bacteria, Mitochondria, and Chloroplasts: Protonmotive Force) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 127. Biomembranes (Part O: Protons and Water: Structure and Translocation) Edited by LESTER PACKER VOLUME 128. Plasma Lipoproteins (Part A: Preparation, Structure, and Molecular Biology) Edited by JERE P. SEGREST AND JOHN J. ALBERS VOLUME 129. Plasma Lipoproteins (Part B: Characterization, Cell Biology, and Metabolism) Edited by JOHN J. ALBERS AND JERE P. SEGREST VOLUME 130. Enzyme Structure (Part K) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 131. Enzyme Structure (Part L) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 132. Immunochemical Techniques (Part J: Phagocytosis and CellMediated Cytotoxicity) Edited by GIOVANNI DI SABATO AND JOHANNES EVERSE VOLUME 133. Bioluminescence and Chemiluminescence (Part B) Edited by MARLENE DELUCA AND WILLIAM D. MCELROY VOLUME 134. Structural and Contractile Proteins (Part C: The Contractile Apparatus and the Cytoskeleton) Edited by RICHARD B. VALLEE VOLUME 135. Immobilized Enzymes and Cells (Part B) Edited by KLAUS MOSBACH VOLUME 136. Immobilized Enzymes and Cells (Part C) Edited by KLAUS MOSBACH VOLUME 137. Immobilized Enzymes and Cells (Part D) Edited by KLAUS MOSBACH VOLUME 138. Complex Carbohydrates (Part E) Edited by VICTOR GINSBURG VOLUME 139. Cellular Regulators (Part A: Calcium- and Calmodulin-Binding Proteins) Edited by ANTHONY R. MEANS AND P. MICHAEL CONN VOLUME 140. Cumulative Subject Index Volumes 102–119, 121–134 VOLUME 141. Cellular Regulators (Part B: Calcium and Lipids) Edited by P. MICHAEL CONN AND ANTHONY R. MEANS VOLUME 142. Metabolism of Aromatic Amino Acids and Amines Edited by SEYMOUR KAUFMAN


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VOLUME 143. Sulfur and Sulfur Amino Acids Edited by WILLIAM B. JAKOBY AND OWEN GRIFFITH VOLUME 144. Structural and Contractile Proteins (Part D: Extracellular Matrix) Edited by LEON W. CUNNINGHAM VOLUME 145. Structural and Contractile Proteins (Part E: Extracellular Matrix) Edited by LEON W. CUNNINGHAM VOLUME 146. Peptide Growth Factors (Part A) Edited by DAVID BARNES AND DAVID A. SIRBASKU VOLUME 147. Peptide Growth Factors (Part B) Edited by DAVID BARNES AND DAVID A. SIRBASKU VOLUME 148. Plant Cell Membranes Edited by LESTER PACKER AND ROLAND DOUCE VOLUME 149. Drug and Enzyme Targeting (Part B) Edited by RALPH GREEN AND KENNETH J. WIDDER VOLUME 150. Immunochemical Techniques (Part K: In Vitro Models of B and T Cell Functions and Lymphoid Cell Receptors) Edited by GIOVANNI DI SABATO VOLUME 151. Molecular Genetics of Mammalian Cells Edited by MICHAEL M. GOTTESMAN VOLUME 152. Guide to Molecular Cloning Techniques Edited by SHELBY L. BERGER AND ALAN R. KIMMEL VOLUME 153. Recombinant DNA (Part D) Edited by RAY WU AND LAWRENCE GROSSMAN VOLUME 154. Recombinant DNA (Part E) Edited by RAY WU AND LAWRENCE GROSSMAN VOLUME 155. Recombinant DNA (Part F) Edited by RAY WU VOLUME 156. Biomembranes (Part P: ATP-Driven Pumps and Related Transport: The Na, K-Pump) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 157. Biomembranes (Part Q: ATP-Driven Pumps and Related Transport: Calcium, Proton, and Potassium Pumps) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 158. Metalloproteins (Part A) Edited by JAMES F. RIORDAN AND BERT L. VALLEE VOLUME 159. Initiation and Termination of Cyclic Nucleotide Action Edited by JACKIE D. CORBIN AND ROGER A. JOHNSON VOLUME 160. Biomass (Part A: Cellulose and Hemicellulose) Edited by WILLIS A. WOOD AND SCOTT T. KELLOGG

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VOLUME 161. Biomass (Part B: Lignin, Pectin, and Chitin) Edited by WILLIS A. WOOD AND SCOTT T. KELLOGG VOLUME 162. Immunochemical Techniques (Part L: Chemotaxis and Inflammation) Edited by GIOVANNI DI SABATO VOLUME 163. Immunochemical Techniques (Part M: Chemotaxis and Inflammation) Edited by GIOVANNI DI SABATO VOLUME 164. Ribosomes Edited by HARRY F. NOLLER, JR., AND KIVIE MOLDAVE VOLUME 165. Microbial Toxins: Tools for Enzymology Edited by SIDNEY HARSHMAN VOLUME 166. Branched-Chain Amino Acids Edited by ROBERT HARRIS AND JOHN R. SOKATCH VOLUME 167. Cyanobacteria Edited by LESTER PACKER AND ALEXANDER N. GLAZER VOLUME 168. Hormone Action (Part K: Neuroendocrine Peptides) Edited by P. MICHAEL CONN VOLUME 169. Platelets: Receptors, Adhesion, Secretion (Part A) Edited by JACEK HAWIGER VOLUME 170. Nucleosomes Edited by PAUL M. WASSARMAN AND ROGER D. KORNBERG VOLUME 171. Biomembranes (Part R: Transport Theory: Cells and Model Membranes) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 172. Biomembranes (Part S: Transport: Membrane Isolation and Characterization) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 173. Biomembranes [Part T: Cellular and Subcellular Transport: Eukaryotic (Nonepithelial) Cells] Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 174. Biomembranes [Part U: Cellular and Subcellular Transport: Eukaryotic (Nonepithelial) Cells] Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 175. Cumulative Subject Index Volumes 135–139, 141–167 VOLUME 176. Nuclear Magnetic Resonance (Part A: Spectral Techniques and Dynamics) Edited by NORMAN J. OPPENHEIMER AND THOMAS L. JAMES VOLUME 177. Nuclear Magnetic Resonance (Part B: Structure and Mechanism) Edited by NORMAN J. OPPENHEIMER AND THOMAS L. JAMES


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VOLUME 178. Antibodies, Antigens, and Molecular Mimicry Edited by JOHN J. LANGONE VOLUME 179. Complex Carbohydrates (Part F) Edited by VICTOR GINSBURG VOLUME 180. RNA Processing (Part A: General Methods) Edited by JAMES E. DAHLBERG AND JOHN N. ABELSON VOLUME 181. RNA Processing (Part B: Specific Methods) Edited by JAMES E. DAHLBERG AND JOHN N. ABELSON VOLUME 182. Guide to Protein Purification Edited by MURRAY P. DEUTSCHER VOLUME 183. Molecular Evolution: Computer Analysis of Protein and Nucleic Acid Sequences Edited by RUSSELL F. DOOLITTLE VOLUME 184. Avidin-Biotin Technology Edited by MEIR WILCHEK AND EDWARD A. BAYER VOLUME 185. Gene Expression Technology Edited by DAVID V. GOEDDEL VOLUME 186. Oxygen Radicals in Biological Systems (Part B: Oxygen Radicals and Antioxidants) Edited by LESTER PACKER AND ALEXANDER N. GLAZER VOLUME 187. Arachidonate Related Lipid Mediators Edited by ROBERT C. MURPHY AND FRANK A. FITZPATRICK VOLUME 188. Hydrocarbons and Methylotrophy Edited by MARY E. LIDSTROM VOLUME 189. Retinoids (Part A: Molecular and Metabolic Aspects) Edited by LESTER PACKER VOLUME 190. Retinoids (Part B: Cell Differentiation and Clinical Applications) Edited by LESTER PACKER VOLUME 191. Biomembranes (Part V: Cellular and Subcellular Transport: Epithelial Cells) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 192. Biomembranes (Part W: Cellular and Subcellular Transport: Epithelial Cells) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 193. Mass Spectrometry Edited by JAMES A. MCCLOSKEY VOLUME 194. Guide to Yeast Genetics and Molecular Biology Edited by CHRISTINE GUTHRIE AND GERALD R. FINK VOLUME 195. Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase Edited by ROGER A. JOHNSON AND JACKIE D. CORBIN

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VOLUME 196. Molecular Motors and the Cytoskeleton Edited by RICHARD B. VALLEE VOLUME 197. Phospholipases Edited by EDWARD A. DENNIS VOLUME 198. Peptide Growth Factors (Part C) Edited by DAVID BARNES, J. P. MATHER, AND GORDON H. SATO VOLUME 199. Cumulative Subject Index Volumes 168–174, 176–194 VOLUME 200. Protein Phosphorylation (Part A: Protein Kinases: Assays, Purification, Antibodies, Functional Analysis, Cloning, and Expression) Edited by TONY HUNTER AND BARTHOLOMEW M. SEFTON VOLUME 201. Protein Phosphorylation (Part B: Analysis of Protein Phosphorylation, Protein Kinase Inhibitors, and Protein Phosphatases) Edited by TONY HUNTER AND BARTHOLOMEW M. SEFTON VOLUME 202. Molecular Design and Modeling: Concepts and Applications (Part A: Proteins, Peptides, and Enzymes) Edited by JOHN J. LANGONE VOLUME 203. Molecular Design and Modeling: Concepts and Applications (Part B: Antibodies and Antigens, Nucleic Acids, Polysaccharides, and Drugs) Edited by JOHN J. LANGONE VOLUME 204. Bacterial Genetic Systems Edited by JEFFREY H. MILLER VOLUME 205. Metallobiochemistry (Part B: Metallothionein and Related Molecules) Edited by JAMES F. RIORDAN AND BERT L. VALLEE VOLUME 206. Cytochrome P450 Edited by MICHAEL R. WATERMAN AND ERIC F. JOHNSON VOLUME 207. Ion Channels Edited by BERNARDO RUDY AND LINDA E. IVERSON VOLUME 208. Protein–DNA Interactions Edited by ROBERT T. SAUER VOLUME 209. Phospholipid Biosynthesis Edited by EDWARD A. DENNIS AND DENNIS E. VANCE VOLUME 210. Numerical Computer Methods Edited by LUDWIG BRAND AND MICHAEL L. JOHNSON VOLUME 211. DNA Structures (Part A: Synthesis and Physical Analysis of DNA) Edited by DAVID M. J. LILLEY AND JAMES E. DAHLBERG VOLUME 212. DNA Structures (Part B: Chemical and Electrophoretic Analysis of DNA) Edited by DAVID M. J. LILLEY AND JAMES E. DAHLBERG


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VOLUME 213. Carotenoids (Part A: Chemistry, Separation, Quantitation, and Antioxidation) Edited by LESTER PACKER VOLUME 214. Carotenoids (Part B: Metabolism, Genetics, and Biosynthesis) Edited by LESTER PACKER VOLUME 215. Platelets: Receptors, Adhesion, Secretion (Part B) Edited by JACEK J. HAWIGER VOLUME 216. Recombinant DNA (Part G) Edited by RAY WU VOLUME 217. Recombinant DNA (Part H) Edited by RAY WU VOLUME 218. Recombinant DNA (Part I) Edited by RAY WU VOLUME 219. Reconstitution of Intracellular Transport Edited by JAMES E. ROTHMAN VOLUME 220. Membrane Fusion Techniques (Part A) ¨ ¨ UNES Edited by NEJAT DUZGU VOLUME 221. Membrane Fusion Techniques (Part B) ¨ ¨ UNES Edited by NEJAT DUZG VOLUME 222. Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part A: Mammalian Blood Coagulation Factors and Inhibitors) Edited by LASZLO LORAND AND KENNETH G. MANN VOLUME 223. Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part B: Complement Activation, Fibrinolysis, and Nonmammalian Blood Coagulation Factors) Edited by LASZLO LORAND AND KENNETH G. MANN VOLUME 224. Molecular Evolution: Producing the Biochemical Data Edited by ELIZABETH ANNE ZIMMER, THOMAS J. WHITE, REBECCA L. CANN, AND ALLAN C. WILSON VOLUME 225. Guide to Techniques in Mouse Development Edited by PAUL M. WASSARMAN AND MELVIN L. DEPAMPHILIS VOLUME 226. Metallobiochemistry (Part C: Spectroscopic and Physical Methods for Probing Metal Ion Environments in Metalloenzymes and Metalloproteins) Edited by JAMES F. RIORDAN AND BERT L. VALLEE VOLUME 227. Metallobiochemistry (Part D: Physical and Spectroscopic Methods for Probing Metal Ion Environments in Metalloproteins) Edited by JAMES F. RIORDAN AND BERT L. VALLEE VOLUME 228. Aqueous Two-Phase Systems ¨ JOHANSSON Edited by HARRY WALTER AND GOTE

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VOLUME 229. Cumulative Subject Index Volumes 195–198, 200–227 VOLUME 230. Guide to Techniques in Glycobiology Edited by WILLIAM J. LENNARZ AND GERALD W. HART VOLUME 231. Hemoglobins (Part B: Biochemical and Analytical Methods) Edited by JOHANNES EVERSE, KIM D. VANDEGRIFF, AND ROBERT M. WINSLOW VOLUME 232. Hemoglobins (Part C: Biophysical Methods) Edited by JOHANNES EVERSE, KIM D. VANDEGRIFF, AND ROBERT M. WINSLOW VOLUME 233. Oxygen Radicals in Biological Systems (Part C) Edited by LESTER PACKER VOLUME 234. Oxygen Radicals in Biological Systems (Part D) Edited by LESTER PACKER VOLUME 235. Bacterial Pathogenesis (Part A: Identification and Regulation of Virulence Factors) Edited by VIRGINIA L. CLARK AND PATRIK M. BAVOIL VOLUME 236. Bacterial Pathogenesis (Part B: Integration of Pathogenic Bacteria with Host Cells) Edited by VIRGINIA L. CLARK AND PATRIK M. BAVOIL VOLUME 237. Heterotrimeric G Proteins Edited by RAVI IYENGAR VOLUME 238. Heterotrimeric G-Protein Effectors Edited by RAVI IYENGAR VOLUME 239. Nuclear Magnetic Resonance (Part C) Edited by THOMAS L. JAMES AND NORMAN J. OPPENHEIMER VOLUME 240. Numerical Computer Methods (Part B) Edited by MICHAEL L. JOHNSON AND LUDWIG BRAND VOLUME 241. Retroviral Proteases Edited by LAWRENCE C. KUO AND JULES A. SHAFER VOLUME 242. Neoglycoconjugates (Part A) Edited by Y. C. LEE AND REIKO T. LEE VOLUME 243. Inorganic Microbial Sulfur Metabolism Edited by HARRY D. PECK, JR., AND JEAN LEGALL VOLUME 244. Proteolytic Enzymes: Serine and Cysteine Peptidases Edited by ALAN J. BARRETT VOLUME 245. Extracellular Matrix Components Edited by E. RUOSLAHTI AND E. ENGVALL VOLUME 246. Biochemical Spectroscopy Edited by KENNETH SAUER VOLUME 247. Neoglycoconjugates (Part B: Biomedical Applications) Edited by Y. C. LEE AND REIKO T. LEE


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VOLUME 248. Proteolytic Enzymes: Aspartic and Metallo Peptidases Edited by ALAN J. BARRETT VOLUME 249. Enzyme Kinetics and Mechanism (Part D: Developments in Enzyme Dynamics) Edited by DANIEL L. PURICH VOLUME 250. Lipid Modifications of Proteins Edited by PATRICK J. CASEY AND JANICE E. BUSS VOLUME 251. Biothiols (Part A: Monothiols and Dithiols, Protein Thiols, and Thiyl Radicals) Edited by LESTER PACKER VOLUME 252. Biothiols (Part B: Glutathione and Thioredoxin; Thiols in Signal Transduction and Gene Regulation) Edited by LESTER PACKER VOLUME 253. Adhesion of Microbial Pathogens Edited by RON J. DOYLE AND ITZHAK OFEK VOLUME 254. Oncogene Techniques Edited by PETER K. VOGT AND INDER M. VERMA VOLUME 255. Small GTPases and Their Regulators (Part A: Ras Family) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 256. Small GTPases and Their Regulators (Part B: Rho Family) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 257. Small GTPases and Their Regulators (Part C: Proteins Involved in Transport) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 258. Redox-Active Amino Acids in Biology Edited by JUDITH P. KLINMAN VOLUME 259. Energetics of Biological Macromolecules Edited by MICHAEL L. JOHNSON AND GARY K. ACKERS VOLUME 260. Mitochondrial Biogenesis and Genetics (Part A) Edited by GIUSEPPE M. ATTARDI AND ANNE CHOMYN VOLUME 261. Nuclear Magnetic Resonance and Nucleic Acids Edited by THOMAS L. JAMES VOLUME 262. DNA Replication Edited by JUDITH L. CAMPBELL VOLUME 263. Plasma Lipoproteins (Part C: Quantitation) Edited by WILLIAM A. BRADLEY, SANDRA H. GIANTURCO, AND JERE P. SEGREST VOLUME 264. Mitochondrial Biogenesis and Genetics (Part B) Edited by GIUSEPPE M. ATTARDI AND ANNE CHOMYN VOLUME 265. Cumulative Subject Index Volumes 228, 230–262

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VOLUME 266. Computer Methods for Macromolecular Sequence Analysis Edited by RUSSELL F. DOOLITTLE VOLUME 267. Combinatorial Chemistry Edited by JOHN N. ABELSON VOLUME 268. Nitric Oxide (Part A: Sources and Detection of NO; NO Synthase) Edited by LESTER PACKER VOLUME 269. Nitric Oxide (Part B: Physiological and Pathological Processes) Edited by LESTER PACKER VOLUME 270. High Resolution Separation and Analysis of Biological Macromolecules (Part A: Fundamentals) Edited by BARRY L. KARGER AND WILLIAM S. HANCOCK VOLUME 271. High Resolution Separation and Analysis of Biological Macromolecules (Part B: Applications) Edited by BARRY L. KARGER AND WILLIAM S. HANCOCK VOLUME 272. Cytochrome P450 (Part B) Edited by ERIC F. JOHNSON AND MICHAEL R. WATERMAN VOLUME 273. RNA Polymerase and Associated Factors (Part A) Edited by SANKAR ADHYA VOLUME 274. RNA Polymerase and Associated Factors (Part B) Edited by SANKAR ADHYA VOLUME 275. Viral Polymerases and Related Proteins Edited by LAWRENCE C. KUO, DAVID B. OLSEN, AND STEVEN S. CARROLL VOLUME 276. Macromolecular Crystallography (Part A) Edited by CHARLES W. CARTER, JR., AND ROBERT M. SWEET VOLUME 277. Macromolecular Crystallography (Part B) Edited by CHARLES W. CARTER, JR., AND ROBERT M. SWEET VOLUME 278. Fluorescence Spectroscopy Edited by LUDWIG BRAND AND MICHAEL L. JOHNSON VOLUME 279. Vitamins and Coenzymes (Part I) Edited by DONALD B. MCCORMICK, JOHN W. SUTTIE, AND CONRAD WAGNER VOLUME 280. Vitamins and Coenzymes (Part J) Edited by DONALD B. MCCORMICK, JOHN W. SUTTIE, AND CONRAD WAGNER VOLUME 281. Vitamins and Coenzymes (Part K) Edited by DONALD B. MCCORMICK, JOHN W. SUTTIE, AND CONRAD WAGNER VOLUME 282. Vitamins and Coenzymes (Part L) Edited by DONALD B. MCCORMICK, JOHN W. SUTTIE, AND CONRAD WAGNER VOLUME 283. Cell Cycle Control Edited by WILLIAM G. DUNPHY VOLUME 284. Lipases (Part A: Biotechnology) Edited by BYRON RUBIN AND EDWARD A. DENNIS


xxxi

VOLUME 285. Cumulative Subject Index Volumes 263, 264, 266–284, 286–289 VOLUME 286. Lipases (Part B: Enzyme Characterization and Utilization) Edited by BYRON RUBIN AND EDWARD A. DENNIS VOLUME 287. Chemokines Edited by RICHARD HORUK VOLUME 288. Chemokine Receptors Edited by RICHARD HORUK VOLUME 289. Solid Phase Peptide Synthesis Edited by GREGG B. FIELDS VOLUME 290. Molecular Chaperones Edited by GEORGE H. LORIMER AND THOMAS BALDWIN VOLUME 291. Caged Compounds Edited by GERARD MARRIOTT VOLUME 292. ABC Transporters: Biochemical, Cellular, and Molecular Aspects Edited by SURESH V. AMBUDKAR AND MICHAEL M. GOTTESMAN VOLUME 293. Ion Channels (Part B) Edited by P. MICHAEL CONN VOLUME 294. Ion Channels (Part C) Edited by P. MICHAEL CONN VOLUME 295. Energetics of Biological Macromolecules (Part B) Edited by GARY K. ACKERS AND MICHAEL L. JOHNSON VOLUME 296. Neurotransmitter Transporters Edited by SUSAN G. AMARA VOLUME 297. Photosynthesis: Molecular Biology of Energy Capture Edited by LEE MCINTOSH VOLUME 298. Molecular Motors and the Cytoskeleton (Part B) Edited by RICHARD B. VALLEE VOLUME 299. Oxidants and Antioxidants (Part A) Edited by LESTER PACKER VOLUME 300. Oxidants and Antioxidants (Part B) Edited by LESTER PACKER VOLUME 301. Nitric Oxide: Biological and Antioxidant Activities (Part C) Edited by LESTER PACKER VOLUME 302. Green Fluorescent Protein Edited by P. MICHAEL CONN VOLUME 303. cDNA Preparation and Display Edited by SHERMAN M. WEISSMAN VOLUME 304. Chromatin Edited by PAUL M. WASSARMAN AND ALAN P. WOLFFE

xxxii


VOLUME 305. Bioluminescence and Chemiluminescence (Part C) Edited by THOMAS O. BALDWIN AND MIRIAM M. ZIEGLER VOLUME 306. Expression of Recombinant Genes in Eukaryotic Systems Edited by JOSEPH C. GLORIOSO AND MARTIN C. SCHMIDT VOLUME 307. Confocal Microscopy Edited by P. MICHAEL CONN VOLUME 308. Enzyme Kinetics and Mechanism (Part E: Energetics of Enzyme Catalysis) Edited by DANIEL L. PURICH AND VERN L. SCHRAMM VOLUME 309. Amyloid, Prions, and Other Protein Aggregates Edited by RONALD WETZEL VOLUME 310. Biofilms Edited by RON J. DOYLE VOLUME 311. Sphingolipid Metabolism and Cell Signaling (Part A) Edited by ALFRED H. MERRILL, JR., AND YUSUF A. HANNUN VOLUME 312. Sphingolipid Metabolism and Cell Signaling (Part B) Edited by ALFRED H. MERRILL, JR., AND YUSUF A. HANNUN VOLUME 313. Antisense Technology (Part A: General Methods, Methods of Delivery, and RNA Studies) Edited by M. IAN PHILLIPS VOLUME 314. Antisense Technology (Part B: Applications) Edited by M. IAN PHILLIPS VOLUME 315. Vertebrate Phototransduction and the Visual Cycle (Part A) Edited by KRZYSZTOF PALCZEWSKI VOLUME 316. Vertebrate Phototransduction and the Visual Cycle (Part B) Edited by KRZYSZTOF PALCZEWSKI VOLUME 317. RNA–Ligand Interactions (Part A: Structural Biology Methods) Edited by DANIEL W. CELANDER AND JOHN N. ABELSON VOLUME 318. RNA–Ligand Interactions (Part B: Molecular Biology Methods) Edited by DANIEL W. CELANDER AND JOHN N. ABELSON VOLUME 319. Singlet Oxygen, UV-A, and Ozone Edited by LESTER PACKER AND HELMUT SIES VOLUME 320. Cumulative Subject Index Volumes 290–319 VOLUME 321. Numerical Computer Methods (Part C) Edited by MICHAEL L. JOHNSON AND LUDWIG BRAND VOLUME 322. Apoptosis Edited by JOHN C. REED VOLUME 323. Energetics of Biological Macromolecules (Part C) Edited by MICHAEL L. JOHNSON AND GARY K. ACKERS


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VOLUME 324. Branched-Chain Amino Acids (Part B) Edited by ROBERT A. HARRIS AND JOHN R. SOKATCH VOLUME 325. Regulators and Effectors of Small GTPases (Part D: Rho Family) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 326. Applications of Chimeric Genes and Hybrid Proteins (Part A: Gene Expression and Protein Purification) Edited by JEREMY THORNER, SCOTT D. EMR, AND JOHN N. ABELSON VOLUME 327. Applications of Chimeric Genes and Hybrid Proteins (Part B: Cell Biology and Physiology) Edited by JEREMY THORNER, SCOTT D. EMR, AND JOHN N. ABELSON VOLUME 328. Applications of Chimeric Genes and Hybrid Proteins (Part C: ProteinProtein Interactions and Genomics) Edited by JEREMY THORNER, SCOTT D. EMR, AND JOHN N. ABELSON VOLUME 329. Regulators and Effectors of Small GTPases (Part E: GTPases Involved in Vesicular Traffic) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 330. Hyperthermophilic Enzymes (Part A) Edited by MICHAEL W. W. ADAMS AND ROBERT M. KELLY VOLUME 331. Hyperthermophilic Enzymes (Part B) Edited by MICHAEL W. W. ADAMS AND ROBERT M. KELLY VOLUME 332. Regulators and Effectors of Small GTPases (Part F: Ras Family I) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 333. Regulators and Effectors of Small GTPases (Part G: Ras Family II) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 334. Hyperthermophilic Enzymes (Part C) Edited by MICHAEL W. W. ADAMS AND ROBERT M. KELLY VOLUME 335. Flavonoids and Other Polyphenols Edited by LESTER PACKER VOLUME 336. Microbial Growth in Biofilms (Part A: Developmental and Molecular Biological Aspects) Edited by RON J. DOYLE VOLUME 337. Microbial Growth in Biofilms (Part B: Special Environments and Physicochemical Aspects) Edited by RON J. DOYLE VOLUME 338. Nuclear Magnetic Resonance of Biological Macromolecules (Part A) ¨ Edited by THOMAS L. JAMES, VOLKER DOTSCH , AND ULI SCHMITZ VOLUME 339. Nuclear Magnetic Resonance of Biological Macromolecules (Part B) ¨ Edited by THOMAS L. JAMES, VOLKER DOTSCH , AND ULI SCHMITZ

xxxiv


VOLUME 340. Drug–Nucleic Acid Interactions Edited by JONATHAN B. CHAIRES AND MICHAEL J. WARING VOLUME 341. Ribonucleases (Part A) Edited by ALLEN W. NICHOLSON VOLUME 342. Ribonucleases (Part B) Edited by ALLEN W. NICHOLSON VOLUME 343. G Protein Pathways (Part A: Receptors) Edited by RAVI IYENGAR AND JOHN D. HILDEBRANDT VOLUME 344. G Protein Pathways (Part B: G Proteins and Their Regulators) Edited by RAVI IYENGAR AND JOHN D. HILDEBRANDT VOLUME 345. G Protein Pathways (Part C: Effector Mechanisms) Edited by RAVI IYENGAR AND JOHN D. HILDEBRANDT VOLUME 346. Gene Therapy Methods Edited by M. IAN PHILLIPS VOLUME 347. Protein Sensors and Reactive Oxygen Species (Part A: Selenoproteins and Thioredoxin) Edited by HELMUT SIES AND LESTER PACKER VOLUME 348. Protein Sensors and Reactive Oxygen Species (Part B: Thiol Enzymes and Proteins) Edited by HELMUT SIES AND LESTER PACKER VOLUME 349. Superoxide Dismutase Edited by LESTER PACKER VOLUME 350. Guide to Yeast Genetics and Molecular and Cell Biology (Part B) Edited by CHRISTINE GUTHRIE AND GERALD R. FINK VOLUME 351. Guide to Yeast Genetics and Molecular and Cell Biology (Part C) Edited by CHRISTINE GUTHRIE AND GERALD R. FINK VOLUME 352. Redox Cell Biology and Genetics (Part A) Edited by CHANDAN K. SEN AND LESTER PACKER VOLUME 353. Redox Cell Biology and Genetics (Part B) Edited by CHANDAN K. SEN AND LESTER PACKER VOLUME 354. Enzyme Kinetics and Mechanisms (Part F: Detection and Characterization of Enzyme Reaction Intermediates) Edited by DANIEL L. PURICH VOLUME 355. Cumulative Subject Index Volumes 321–354 (in preparation) VOLUME 356. Laser Capture Microscopy and Microdissection Edited by P. MICHAEL CONN VOLUME 357. Cytochrome P450 (Part C) (in preparation) Edited by ERIC F. JOHNSON AND MICHAEL R. WATERMAN


xxxv

VOLUME 358. Bacterial Pathogenesis (Part C: Identification, Regulation, and Function of Virulence Factors) (in preparation) Edited by VIRGINIA L. CLARK AND PATRIK M. BAVOIL VOLUME 359. Nitric Oxide (Part D) (in preparation) Edited by ENRIQUE CADENAS AND LESTER PACKER VOLUME 360. Biophotonics (Part A) (in preparation) Edited by GERARD MARRIOTT AND IAN PARKER VOLUME 361. Biophotonics (Part B) (in preparation) Edited by GERARD MARRIOTT AND IAN PARKER


Section I Basic Principles


[1]

COMPARISON OF CURRENT EQUIPMENT

3

[1] Comparison of Current Equipment By ANDA CORNEA and ALISON MUNGENAST Introduction Some of the most exciting new developments in biomedical research, such as DNA microarrays and proteomics, depend on the isolation of single cells or pure populations of cells with specific phenotypes. Several microscopic techniques are currently used for microdissection: suction of a cell content through micron-size glass pipettes, dissection using a piezo-activated metal knife and suction through a glass pipette (PPMD by Brinkmann), and dissection using lasers. Because of the high energy concentrated into a small area, the easy control of the beam position, and the lack of direct contact with the material to be dissected, lasers provide the best option for easy-to-use, large-scale microdissections. There are currently three commercially available systems designed specifically for laser capture microdissection: PixCell by Arcturus (Mountain View, CA), PALM by P.A.L.M. Mikrolaser Technologie (Wolfratshousen, Germany), and the Leica AS LMD by Leica (Heidelberg, Germany). The PixCell originated in a Cooperative Research and Development Agreement between the National Institutes of Health, the National Cancer Institute, and the National Institute for Child and Human Development and is now manufactured and marketed by Arcturus. The system uses an IR laser focused through a microscope objective to heat a plastic film placed above a section of tissue. The plastic melts temporarily in the small area irradiated and penetrates the tissue. When the laser beam is turned off, the plastic solidifies and forms bonds with the tissue it has penetrated. When the plastic sheet is removed, the tissue bonded to the plastic is removed as well and thereby isolated or dissected from the rest (Fig. 1). The dissected material may then be processed for the isolation of RNA, DNA, or proteins (Emmert-Buck et al.1 ). The PALM system uses an N2 laser with 336 nm wavelength. The laser, also focused through an inverted microscope objective, has enough energy to ablate tissue or cells that are in focus. Ablation, which destroys chemical bonds within a tissue by a mechanism not fully understood, may remove undesirable cells or groups of cells and isolate a region of interest. When the laser is slightly defocused, its energy may be used to catapult the dissected material up to where it may be collected into a cap and stored or immediately used for the isolation of RNA, DNA, 1

M. R. Emmert-Buck, R. F. Bonner, P. D. Smith, R. F. Chuaqui, Z. Zuang, S. R. Goldstein, R. A. Weiss, and L. A. Liotta, Science 274, 998 (1996).

METHODS IN ENZYMOLOGY, VOL. 356

Copyright 2002, Elsevier Science (USA). All rights reserved. 0076-6879/02 $35.00

4

[1]

BASIC PRINCIPLES

Laser beam Plastic film Tissue section Glass slide

A

Dissected cell

B

Plastic film

Tissue section Glass slide

FIG. 1. Principle of laser capture microscopy used by Arcturus. (A) A thin plastic film is lowered over the specimen to be dissected. An IR laser beam focus through a microscope objective illuminates a small area on the plastic causing it to melt locally and penetrate the tissue. (B) When the laser is interrupted, the plastic solidifies forming bonds with the underlying tissue. When the plastic is removed, the attached tissue is isolated from the rest of the specimen.

or proteins (Schutze and Lahr2 ). This process, termed laser pressure catapulting, is patented by P.A.L.M. (Fig. 2). The Leica AS LMD, more recently introduced as the third generation of laser microdissection systems, uses a pulsed UV laser similar to the PALM on an upright microscope. The laser beam may be moved with a software-controlled mirror system to select cells to be ablated or to isolate the area to be dissected. The dissected material is allowed to fall by gravity into a cap and may thereafter be used for isolating proteins or genetic material (Fig. 3). There are several parameters that may be used to characterize a laser microdissection system. Among them the most important are the resolution and the specificity of dissection and the integrity of the dissected material. Very important also are the ease of use, the reliability of the instrument, and the quality of service and support. Resolution The smallest area that can be isolated from the rest of a tissue by laser microdissection is related to the size of the laser beam and therefore depends on the numerical aperture (NA) of the objective used for dissection and on the wavelength of light used. The NA of an objective is defined as the sine of the collection 2

K. Schute and G. Lahr, Nat. Biotechnol. 16, 737 (1998).

[1]


5

Tissue section Plastic film Glass slide

A Laser beam (ablating)

B Laser beam (catapulting)

C Laser beam (catapulting) FIG. 2. Principle of the PALM laser microdissection. (A) A UV laser beam focused by the objective of an inverted microscope cuts a contour around the area to be dissected. (B) The laser is defocused and positioned within the selected area. (C) The laser pressure is used to lift the dissected sample into a collecting cap. This process, named laser pressure catapulting, is patented by P.A.L.M.

angle multiplied by the refractive index of the immersion medium. Most common configurations for laser microdissection use dry objectives, which somewhat limits the NA. The most stringent limitation comes, however, from the maximum collection angle that can be used. For limited lens diameters, a large angle imposes a short working distance. In most cases, specimens to be dissected are mounted on standard microscope glass slides with approximately 1 mm thickness. For the PALM and the Leica AS LMD systems, the light exiting the objective needs to cross this distance in order to be focused on the tissue. This requires long working distance objectives that necessarily have a lower NA and consequently a wider laser beam waist. For cases when the thinnest possible cuts are required, higher NA objectives may be used if the specimen is mounted on a thin glass coverslip, making possible the use of short working distance objectives with high NA. The main inconvenience in this case is the fragility of these coverslips, as all steps prior to dissection, including collection of sections, fixing, staining, transport, and mounting on the microscope stage, have to be done with extra care. In the PixCell, light leaving the objective passes through the collecting cap and that also limits the minimum usable working distance and consequently the NA of

6

[1]

BASIC PRINCIPLES

Laser beam (ablating) Glass slide Plastic film Tissue section

A

Laser beam (ablating)

B

C Dissected cell FIG. 3. Principle of the Leica AS LMD microdissection. (A) A laser beam similar to the one used by PALM is concentrated by the objective of an upright microscope. The specimen, mounted on a PEN membrane, is mounted upside down. (B) A contour is cut through the membrane and tissue around the area to be dissected by moving the laser beam and not the stage. (C) The dissected area, isolated from the rest of the specimen, falls into a collecting cap positioned under the specimen.

the objective. In this case, the user does not have the option to mount specimens on coverslips; the only improvement could come from redesigning the collection caps. The size of the beam waist depends on the wavelength of light used. The infrared light used by PixCell will give a larger beam waist than the UV light used by the PALM and Leica AS LMD for the same objective used. The demands for the smallest possible beam waist, however, are different for the two classes of microdissection instruments, as the dissection technique is different. For the PixCell, the beam waist gives the minimum dissected area. The minimum value cited by the manufacturer is 7.5 µm. This is about the size of a cell, or smaller than many cells. It can be argued that a smaller size is hardly necessary, as in most cases laser microdissection is used to isolate single cells or larger numbers of identical cells and, in this case, the intent is to collect as much material per cell as possible. The PALM and Leica AS LMD use the laser to ablate a contour line around a single cell and therefore the width of the cut is expected to be much smaller. The minimum cut sizes quoted for the PALM and Leica AS LMD were less than 1 µm and 2.5 µm, respectively.

[1]


7

Specificity and Integrity In all systems the material removed may include more than the specific area marked for dissection. In the case of the PixCell, the bond between the plastic and the tissue must be stronger than the bond between tissue and slide in order to allow removal of the cell or tissue part marked for dissection. Uncharged and unsubbed slides work well because the tissue adheres more loosely to the slides. This presents a problem, however, when the user desires to process the tissue beyond simple staining, such as using immunohistochemistry, as sections may be lost during the procedure. The bonds within the tissue, dependent on tissue type and fixation, may also overcome in some cases the attachment to the slide, and extra material will be removed contaminating the purity of the dissected sample (Fig. 4). For some of the collecting caps, the most frequently used ones, the plastic film touches the section of tissue to be dissected and may remove material that randomly adhered to it. This is, however, overcome in the more newly designed “CapSure” caps in which only the melted film touches the tissue. Sticky “prep strips” are also provided which can reduce contamination.

FIG. 4. Positive selection of cells to be dissected with PixCell laser capture microscope. (A) Brain section in which cells selected for dissection appear in a lighter color after the plastic transfer film was attached by melting. (B) Cells removed with the film. Arrows indicate cells for which nuclei failed to transfer. Arrowheads indicate dissection that removed extra material. Note the clarity of quality control.

8

BASIC PRINCIPLES

[1]

FIG. 5. Negative selection of area to be selected by a PALM laser microdissection microscope. (A) A freehand line is drawn on the screen over the image. (B) The laser cuts the PEN membrane and ablates the tissue along that line. (C) The tissue enclosed by the cut is catapulted using laser pressure. (D) Sample remaining after positive selection of single cells in a different tissue section mounted directly on a glass slide.

The laser ablation used in the PALM and Leica LDM is aimed at circumventing this contamination problem by destroying the tissue around the cell or the region of interest that can then be collected free of neighboring contamination (Fig. 5). The process of ablation itself is not well understood and the destruction of chemical bonds may not be complete. Particles of the material targeted for ablation may be seen, while cutting, landing on the cell or region to be dissected suggesting a potential for contamination. Cells adjacent to the cell of interest cannot be collected as well with this process, as they are ablated by the laser beam. With the Arcturus, however, it is possible to collect material from adjacent cells. The integrity of the material dissected may be an issue as in all cases the sample is irradiated with laser light that has the potential to alter chemical bonds by either destroying them or causing cross-linking. This is particularly an issue for the UV lasers even though the wavelength is slightly larger than the main absorption peak for proteins and nucleic acids. The high temperature created by the IR laser in the PixCell system, necessary for the melting of the film, may also degrade the dissected material. There is at this time a large body of literature suggesting that adequate RNA may be quantitatively isolated by both technologies and not much published evidence to the contrary. Ease of Use There are many steps involved in laser capture microdissection that may affect the ease of use. The type of slides required, demands on sectioning and processing of the tissue, visualization and selection of the region to be selected, and dissection

[1]


9

and collection of the dissected material are all variables that may make the technique easy and straightforward or complicated and awkward. Regular microscope slides may be used in all systems, albeit with suboptimal results. For the PALM and the Leica DML it is recommended that a thin PEN (polyethylene naphthalate) membrane be mounted between the slide and the tissue. The membrane may be cut by the ablation laser around the area targeted for dissection and then catapulted in the PALM or let drop in the Leica, preserving the integrity of the cells attached to it. Dissected pieces of tissue may be visualized after capture in the collecting cap only if the underlying membrane kept the structure intact. If cells are catapulted directly from a slide, the material will be pulverized and it will be impossible to assess the efficiency of capture by visualization. PEN coated slides may be purchased but are relatively expensive. Preparing them, however, may be very time consuming. We have also found that the membranes tear easily during immunohistochemical processing. For the PixCell, the tissue may be mounted directly on a glass slide. The dissected cells are then collected on a plastic film attached to the collecting caps and can be easily visualized. For all systems, the efficiency of dissection and collection is critically dependent on tissue preparation as well as on environmental factors.

Sample Preparation Arcturus provides a number of protocols for sample preparation that must be strictly followed in order to obtain a good dissection. The thickness of the section, method of fixation of the tissue, and staining are restricted. Specimens need to be perfectly dry to adhere to the transfer film. The PALM and the Leica systems rely largely on the ablation of the PEN foil for dissection and therefore offer more freedom for the preparation of the specimen. Tissue sections may be much thicker and various fixations may be used. The PALM may even isolate live cells. Not everything works, however, and the greater freedom in sample preparation comes with the necessity for users to optimize their own protocols.

Environment Humidity in the specimen, due either to incomplete drying or to absorption of air vapors, makes dissection in the PixCell impossible. Samples and instrument need to be kept in a dry environment. Humidity between the slide and the membrane prevents proper membrane cutting in the PALM. Lack of humidity, however, contributes to increased electrostatic forces that compete with gravity and affect collection in the Leica AS LMD system. In this case, the instrument should be kept in a high-humidity room free of air drafts.

10

BASIC PRINCIPLES

[1]

Control of Stage Movement The control of the stage and selection of areas for dissection may strongly affect the ease of use. The PixCell uses a joystick manual control of the stage movement. The stage is moved so that each intended target is brought into the middle of the field of view, under the laser beam, and dissected, before moving to the next target and so on. If areas larger than can be covered by a single beam are desired, multiple laser shots need to be fired as the stage is moved. This requires skill and concentration, as any misfire can contaminate the whole sample. The PALM uses computer-controlled stage motors that can move the stage along a predrawn path. This is particularly convenient when large areas of irregular shapes are dissected. A freehand drawing tool allows the operator to outline the target area, and then the stage moves as the laser fires and cuts the underlying membrane along the chosen path. The whole area is then catapulted into the collecting cap. When several isolated cells are collected into the same cap, they can each be outlined first, then the system isolates them one by one, automatically. The Leica AS LMD uses a computer-controlled mirror that moves the laser beam along a path also preselected by a freehand drawing tool. Cutting may be in this case much faster than for the PALM, as the stage is immobile and the laser beam is moved faster.

Service A major factor in the satisfaction with any of the instruments chosen is the reliability and speed and quality of service. Arcturus has the longest history of operation and, at least in the United States, has an excellent record of reliability. The IR laser it uses is designed for a longer lifetime than the UV laser used by the PALM and Leica estimated at 2 years or 2,000,000 pulses. The PALM, now marketed and supported by Zeiss, and Leica are expanding their operations in the United States, relying for applications and service on the preexisting networks of the two respective companies. We have tested all three systems in an attempt to find the one that best fits most of our needs. Our experience showed that for all three systems, sample preparation and environmental factors are critical for good dissection. Specimens freshly prepared according to the Arcturus-suggested protocols could be very easily dissected. Samples insufficiently dried or stored without desiccation could not be dissected. Specimens prepared for demonstration by the PALM could be easily dissected and catapulted. With a specimen that we prepared with membrane coated slides provided by PALM which maintained a little humidity, the membrane could not be cut reliably even with the highest setting for the laser energy. When the tissue was mounted directly on a glass slide without a membrane, individual cells could be easily catapulted into the collecting cap.

[1]


11

Dissection using the Leica AS LMD system was less sensitive to sample preparation. Tissue sections were mounted in this case on a thin PEN membrane held by a plastic frame the size of a microscope slide that was easy to dry. Dissected samples, however, were subjected to strong electrostatic attraction from the many charged plastic and metal surfaces close by, allowing only few of them to be collected in the caps. Air humidity in this case is helpful in reducing the electrostatic charges around the membrane. Shooting the slide with an electrostatic gun also helped to some degree. The ability to visually inspect the dissected material was excellent for the Arcturus system. The images of the remaining and dissected material, acquired with the same objective, match each other perfectly and may be added to reconstitute the image before the dissection. In the case of the PALM, larger areas dissected could be visually inspected with relative ease, albeit with a lower magnification objective. Cells mounted directly on the glass slide, dissected by catapulting, are practically pulverized and therefore cannot be seen. Dissected cells could not be seen in the Leica AS LMD. For each instrument we tested the presence and integrity of RNA in the collecting caps. Groups of cells or single cells were captured with each microscope. Cells were snap-frozen immediately after capture. RNA was extracted using the Arcturus PicoPure RNA Isolation kit following the Arcturus protocol. Extracted RNA was dried down to 1.6 µl in a vacuum centrifuge. The following components were added to each sample: 1.7 µl Invitrogen 1st Strand Buffer, 0.5 µl RNasin (Promega), 1.7 µl 0.1 M DTT (Invitrogen), 1 µl dNTPs (Promega), 100 ng Random Hexamers (Invitrogen), and 1 µl Superscript II Reverse Transcriptase (Invitrogen) to a final volume of 8.5 µl. The samples were incubated at 42◦ for 90 min.

FIG. 6. All captured samples contain intact mRNA. Laser-captured samples were subjected to RNA extraction with the Arcturus PicoPure kit and reverse transcription with the GIBCO-BRL Superscript II enzyme. Two rounds of PCR were performed with nested primers recognizing cyclophilin, a housekeeping gene. Lanes 1 and 2 contain a sample isolated with the PALM equipment, lanes 3 through 8 contain samples isolated with the Leica AS LMD, and lanes 9 and 10 contain a sample isolated with the Arcturus PixCell system. Lanes 11 and 12 contain PCR products from control rat hypothalamic mRNA. Lanes 1, 3, 5, 7, 9, and 11 represent the first round of nested PCR using outer primers. Lanes 2, 4, 6, 8, 10, and 12 represent the final PCR product after a second round of PCR with inner primers.

12

[2]

BASIC PRINCIPLES TABLE I COMPARISON OF CURRENT EQUIPMENT PixCell II

PALM

Leica LMD

+++ ++ + ++ +++ +++ ++ +++ ++ +++ ++

+ +++ +++ +++ ++ ++ +++ + ++ +++ +

+ +++ ++ +++ ++ ++ +++

Laser lifetime Resolution Versatility Sample preparation Sample preparation protocols Ease of use—single cells Ease of use—larger areas Visualization of dissected sample Sample recovery RNA integrity Price

+ +++ ++

After the reverse transcription reaction, 1 µl from each sample was subjected to two 35-cycle rounds of PCR with nested primers recognizing cyclophilin, a housekeeping gene. The PCR products from both rounds were electrophoresed on a 2% agarose gel (Fig. 6). Our experience with the instruments tested is summarized in Table I. Addendum: Useful Sites http://www.arctur.com/ http://www.palm.spacenet.de/ http://www.leica-microsystems.com/

[2] Laser Capture Microdissection and Its Applications in Genomics and Proteomics By JAMES L. WITTLIFF and MARK G. ERLANDER Background Human tissue collection, handling, and analyses present specific problems for clinically reliable genomic and proteomic testing unlike studies with animal tissues or homogeneous cell lines grown in culture. For example, determinations of levels of clinically relevant analytes in tissue biopsies, used as markers for detection, diagnosis, prognosis, or therapeutic response of a cancer patient, are performed



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either biochemically or by immunohistochemistry currently (e.g., Wittliff et al.1 ). If the analyte (e.g., estrogen receptor, HER-2/neu oncoprotein) is measured biochemically, a tissue specimen consisting of a heterogeneous cell population is homogenized and the final concentration of the analyte extracted from the cancer cells is “diluted” by the contribution of other proteins released from noncancerous cells (e.g., epithelium, histiocytes, macrophages, and connective tissue cells). Therefore, an underestimate of the analyte concentration is likely to be determined compromising the appropriate cutoff value between disease and normal states. While certain tumor markers in tissue biopsies have well served the clinical management of cancer patients (e.g., estrogen receptors in the selection of tamoxifen-responsive breast cancer1 ), many questions of analyte expression in normal and neoplastic cells remain. Likewise, immunohistochemistry is used to measure certain proteins in cancer tissue sections for clinical application in spite of reports indicating the results are often highly operator and antibody dependent and, at best, semiquantitative (e.g., Igarashi et al.2 ). As Wittliff1 and Cole et al.3 have noted, collection and processing of human tissue biopsies have focused on their clinical purpose (e.g., diagnosis, staging, prognosis, therapy selection) with little emphasis on sampling and cryopreservation for sophisticated genomic (e.g., microarrays) and proteomic analyses (e.g., protein chips). The obvious problem of cellular heterogeneity in the tissue section, which may result in misleading or confusing molecular findings,3 complicates these issues. Therefore, a reproducible method for obtaining homogeneous cell populations from normal tissue or from cancer biopsies was required in order to obtain accurate information from molecular analyses. Laser capture microdissection (LCM) was initially conceived by a team of investigators at the National Institutes of Health, led by Lance Liotta, Robert Bonner, and Michael Emmert-Buck, to address this need.4,5 LCM provides a rapid and direct method for procuring homogeneous subpopulations of cells or complex structures for biochemical and molecular biological analyses. Arcturus Engineering, Inc. (Mountain View, CA) developed the first commercial LCM instrument, made available in 1997, in collaboration with the NIH group as part of a Cooperative Research and Development Agreement (CRADA). 1

J. L. Wittliff, R. Pasic, and K. I. Bland, in “The Breast: Comprehensive Management of Benign and Malignant Diseases” (K. I. Bland and E. M. Copeland III, eds.), p. 458. W. B. Saunders Co., Philadelphia, 1998. 2 H. Igarashi, H. Sugimura, K. Maruyama, Y. Kitayama, I. Ohta, M. Suzuki, M. Tanaka, Y. Dobashi, and I. Kino, APMIS 102, 295 (1994). 3 K. A. Cole, D. B. Krizman, and M. R. Emmert-Buck, Nat. Genet. 21, 38 (1999). 4 M. R. Emmert-Buck, R. F. Bonner, P. D. Smith, R. F. Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, and L. A. Liotta, Science 274, 998 (1996). 5 R. F. Bonner, M. R. Emmert-Buck, K. Cole, T. Pohida, R. Chuaqui, S. Goldstein, and L. A. Liotta, Science 278, 1481 (1997).

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FIG. 1. Components of a laser capture microdissection instrument. The LCM station integrates a research grade inverted microscope, a low-power infrared laser, a joystick-controlled stage, and a custom CapSure LCM cap handling mechanism (cassette module and placement arm). The LCM employs a video camera connected to an image archiving unit (not shown) for annotation, storage, and review of the microdissection process.

Laser Capture Microdissection Instrumentation LCM represents a major advancement in nondestructive cell sampling technology that can be applied to genomic and proteomic studies. Studies conducted in our laboratories utilize the PixCell II LCM System (Arcturus Engineering, Inc.) composed of the LCM instrument with fluorescence microscopy, the CapSure Transfer Film Carrier, and the PixCell II Image Archiving Workstation (Fig. 1). Briefly, the LCM station integrates a research-grade inverted microscope, a lowpower infrared laser, a joystick-controlled stage, and a custom CapSure LCM cap handling mechanism with a video monitor and controller. Protocol for Processing Human Tissue Specimens To evaluate differences between normal and diseased cells, one must first isolate the cells or structures by LCM (Fig. 2) and extract them independently for DNA, RNA, or protein analyses.6,7 Proper tissue procurement, specimen handling, and cryopreservation are essential for the collection of quality information from these analyses.1 Briefly, biopsy specimens should be excised expeditiously and without trauma during the surgical procedure. Specimens must be chilled on ice, 6

N. L. Simone, R. F. Bonner, J. W. Gillespie, M. R. Emmert-Buck, and L. A. Liotta, Trends Genet. 14, 272 (1998). 7 J. L. Wittliff, S. T. Kunitake, S. S. Chu, and J. C. Travis, J. Clin. Ligand Assay 23, 66 (2000).

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FIG. 2. Sequence for LCM procurement of cells from a complex tissue section. After tissue collection, fixation, staining, and dehydration as described in the text, cells of interest are located and the CapSure optically transparent cap is placed on the tissue. A laser pulse releases the cell from surrounding structures transferring it to the thermoplastic film. The intact cell bound to the CapSure device is lifted and placed onto a standard 500 µl microcentrifuge tube for subsequent extraction and analysis.

and then well trimmed of necrotic tissue, leaving normal tissue present with the lesion in question. The tissue specimen should either be frozen on dry ice in the pathology suite within 20–30 min of collection or rapidly transported chilled in a petri dish or plastic bag immersed in ice prior to cryopreservation and frozen section preparation in the LCM laboratory to retain the biological integrity of macromolecules. Any procedure avoiding RNase contact is desirable. It is preferable to freeze the specimen immediately on dry ice after collection at the time of frozen section diagnosis if studies requiring RNA are to be conducted. With the advent of LCM and sensitive technologies of genomics and proteomics requiring nondestructive isolation of pure cell populations, new surgical pathology

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approaches and methods must be developed as recommended by Cole et al.3 and Wittliff et al.7 Specimens are processed according to accepted biohazard policies in clean rooms prepared to reduce RNase contamination. Specimens are frozen in an optimum cutting temperature compound (TissueTek OCT medium, VWR Scientific Products Corp.) and stored at −86◦ until LCM is performed. At that time, frozen sections are collected on sterile microscope slides without a coverslip and retained frozen by being placed on a flat surface of dry ice to preserve labile macromolecules. We recommend that glass slides without coatings (uncharged) be used to enhance LCM of selected cells. Frozen tissue biopsies or tissue sections collected on slides and stored in sterile plastic slide holders may be shipped to a distant laboratory for LCM analyses if the specimens are retained on dry ice during transfer. Preservation of the biological integrity of the biopsy tissue prior to arrival in the LCM laboratory is the shared responsibility of the pathologist and the surgeon, if proteomic and genomic analyses are to become routine clinical tests. In addition, sections of the tissue procured must be representative of the lesion. Fixation, Staining, and Dehydration Frozen sections mounted on uncoated glass slides are handled according to the following procedures depending on the type of staining reagent used. The intercalating dye, TO-PRO-3 (Molecular Probes, Inc., Eugene, OR), which binds tightly to double-stranded nucleic acids and exhibits a peak fluorescence at 661 nm, has been used to assess the integrity of DNA in LCM procured cells and structures.7 TO-PRO-3 Staining Protocol (1) Place frozen section in 70% ethanol for 1 min, (2) transfer to PBS for 30 sec, (3) place slide in tray and stain with 10 µM TO-PRO-3 for 2 min, (4) transfer to PBS for 2 min, (5) transfer to deionized water for 30 sec, (6) transfer to 70% ethanol for 30 sec, (7) transfer to 95% ethanol for 30 sec, (8) transfer to 100% ethanol for 30 sec, (9) transfer to xylene for 5 min, and (10) air dry for 20 min; store desiccated. Phosphate-buffered saline (PBS) and deionized water are used in all wash steps. All of the steps utilizing ethanol employed ethyl alcohol UPS (Aaper Alcohol and Chemical Co., Shelbyville, KY). H&E Staining Protocol (1) Place frozen section in 70% ethanol for 1 min, (2) transfer to hematoxylin Gill No. 3 (Sigma Diagnostics, St. Louis, MO) for 30 sec, (3) transfer to RNasefree water for 30 sec, (4) transfer to bluing agent (ThermoShandon, Pittsburgh, PA) for 30 sec, (5) transfer to 70% ethanol for 30 sec, (6) transfer to 70% alcohol-based eosin Y alcoholic (ThermoShandon, Pittsburgh, PA) for 30 sec, (7) transfer to 70%

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FIG. 3. Representative images of human breast carcinomas stained with H & E. (A) Typical human breast carcinoma showing infiltration of cancer cells into stromal elements; (B) breast carcinoma adjacent to a large population of inflammatory cells; (C) tissue specimen of DCIS with protein secretions; (D) breast carcinoma biopsy exhibiting freezing artifact.

ethanol for 30 sec, (8) transfer to 95% ethanol for 30 sec, (9) transfer to 100% ethanol for 30 sec, (10) transfer to xylene GR/ACS (EM Science, Gibbstown, NJ) for 5 min, and (11) air dry for 20 min; store desiccated. Desiccate only if slide will not be used for RNA extraction. Tissue slides to be used for total RNA extraction and gene expression profiling must be used within 1–2 hr for LCM procurement of cells. Prior to LCM, we evaluate the structural status of the frozen tissue biopsy after sectioning and H & E staining (Fig. 3). As illustrated in Fig. 3A, the section indicates the biopsy is acceptable for proceeding with LCM and gene expression profiles. The section shown in Fig. 3B also indicates an acceptable specimen but considerable caution must be exercised to avoid removing unwanted inflammatory cells with carcinoma cells. The tissue section shown in Fig. 3C illustrates that the specimen received in the laboratory contained considerable areas of DCIS although

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the diagnosis indicated invasive ductal carcinoma. This illustrates the value of pathology confirmation on the portion of the tissue biopsy received in the LCM laboratory. Finally, the tissue section shown in Fig. 3D depicts significant freezing artifact indicating the biopsy was unsatisfactory for LCM and gene expression profiling. Prior to LCM, other types of tissue preparations were utilized. These include either formalin-fixed or alcohol-fixed sections that are paraffin-embedded, as well as cytospin preparations of cells from blood or ascites fluids. Fixation conditions are dictated by the nature of the antigen of interest. Precipitative reagents such as acetone and methanol are used for intracellular antigens while cross-linking fixatives such as glutaraldehyde or paraformaldehyde are used for cell-surface antigens.8 Immunohistochemistry of protein analytes has been performed to guide cell selections by LCM (e.g., Fend et al.9 ). We routinely perform immunohistochemistry of clinically relevant analytes such as estrogen and progestin receptors, EGF receptors, and HER-2/neu oncoprotein to direct the procurement of cells expressing particular tumor markers.1 Steps in Laser Capture Microdissection The sequence of tissue collection, cell procurement by LCM, and macromolecular extraction is depicted in Fig. 4. Avoid the presence of moisture (e.g., frost, fingertips, breath, room humidity) during all steps prior to RNA extraction. Because our LCM laboratories are used for proteomic and gene expression studies, all procedures are conducted under RNase-free conditions, including cleaning of the stage and related areas of the LCM instrument and surrounding bench with RNase AWAY (Molecular BioProducts, San Diego, CA). Gloves and lab coats are worn at all times. Prior to performing LCM, the joystick should be positioned perpendicular to the bench and the CapSure LCM cap should be placed over the tissue under examination. The operator locates the cell or structure to be microdissected from the tissue section by viewing the histology on the monitor of the PixCell II LCM System.7 After test firing the IR laser in an area devoid of cells and observing the features of the melted plastic ring, the settings for power and duration are adjusted to obtain the desired spot size. Typically if one is using CapSure HS LCM caps, the following adjustments are suggested: spot size of 7.5 µm (power setting = 65–75 mW; duration setting = 650–750 µs), spot size of 15 µm (power setting = 35–45 mW; duration setting = 2.5–3.0 ms), spot size of 30 µm (power 8

S.-R. Shi, J. Gu, and C. R. Taylor, “Antigen Retrieval Techniques: Immunohistochemistry and Molecular Morphology.” Eaton Publishing, Natick, MA, 2000. 9 F. Fend, M. R. Emmert-Buck, R. Chuaqui, K. Cole, J. Lee, L. A. Liotta, and M. Raffeld, Am. J. Pathol. 154, 61 (1999).

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FIG. 4. Simple four-step process to capture cells and recover macromolecules. After location of the cells of interest, a CapSure or CapSure HS LCM cap is placed over the target area. Pulsing the laser through the cap activates the thermoplastic film to form a thin protrusion that bridges the gap between the cap and tissue and adheres to the target cell. Lifting of the cap removes the target cell(s) now attached to the cap. Macromolecules may be extracted from 200–1000 cells using the ExtracSure Sample Extraction Device which accommodates small volumes.

setting = 45–55 mW; duration setting = 6.0–7.0 ms). If CapSure LCM caps are employed, the following adjustments are suggested: spot size of 7.5 µm (power setting = 40–50 mW; duration setting = 550–650 µs), spot size of 15 µm (power setting = 30–40 mW; duration setting = 1.5–2.0 ms), spot size of 30 µm (power setting = 25–35 mW; duration setting = 5.0–6.0 ms). In order to correctly return to the initial area of cell capture for making photo records of

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“After” and “Cap” images (Fig. 5), the authors “mark” the region by placing several spots on areas devoid of cells adjacent to the cells of interest. This is particularly helpful when collecting multiple caps of cells from distant regions in the same tissue section for later comparison of proteomic and genomic analyses. Using the image archiving unit, characteristics of the tissue section are recorded before and after LCM, as well as those of the cells procured on each cap (Fig. 5). The cells or structures are microdissected after firing the IR laser and lifting the CapSure LCM cap with the intact cells collected on the transfer film. The CapSure and CapSure HS consist of a proprietary thermoplastic polymer film hermetically sealed to the bottom of a precision optical grade plastic cap. In certain experiments requiring extraction of small numbers of cells (200–1000) in low microliter volumes, we utilize the ExtracSure Sample Extraction Device (Arcturus) and the CapSure HS LCM caps for efficient removal of total RNA. The CapSure LCM caps containing the cells fit directly onto standard reagent tubes (500 µl Eppendorf) in preparation for cell extraction. Typically, 1–6 ng of total RNA may be extracted in this manner using Buffer RLT (Qiagen, Valencia, CA). The transfer process does not damage the captured cells or the surrounding cells remaining on the slide containing the original tissue preparation (Fig. 5). Usually there is no undesirable cellular contamination since the IR laser beam may be focused between 7.5 and 30 µm providing accurate selection. If necessary, we employ the CapSure pads to remove debris (e.g., stromal elements) from the CapSure LCM caps prior to extraction. Forces involved in an efficient LCM manipulation include (a) those between tissue and slide, (b) those between tissue and activated film, (c) tissue–tissue interactive forces, and (d) the force between tissue and inactivated film. The dynamics of the IR focusing and the melting properties of the thermoplastic transfer film on the CapSure LCM caps are optimized with those of cells in 5- to 10-µm tissue sections. After collection of cells on the CapSure LCM cap, macromolecules are extracted using a variety of procedures depending on whether the analyses are focused on DNA, RNA, or protein, as described in other chapters of this volume. Gene expression as measured by analyses of mRNA provides an understanding of the manner in which normal cells respond to endocrine changes, malignant transformation, and environmental insults.6,7,10–13 Determination of the level of gene 10

A. Glasow, A. Haidan, U. Hilbers, M. Breidert, J. Gillespie, W. A. Scherbaum, G. P. Chrousos, and S. R. Bornstein, J. Clin. Endocrin. Metab. 83, 4459 (1998). 11 L. Luo, R. C. Salunga, H. Guo, A. Bhittner, K. C. Joy, J. E. Galindo, H. Xiao, K. E. Rogers, J. S. Wan, M. R. Jackson, and M. G. Erlander, Nat. Med. 5, 117 (1999). 12 D. C. Sgroi, S. Teng, G. Robinson, R. LeVangie, J. R. Hudson, Jr., and A. G. Elkahloun, Cancer Res. 59, 5656 (1999). 13 S. M. Goldsworthy, P. S. Stockton, C. S. Trempus, J. F. Foley, and R. R. Maronpot, Mol. Carcinog. 25, 86 (1999).

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FIG. 5. Representative collection of human breast cancer cells by LCM. Three different regions of the same breast carcinoma biopsy are shown in the top three panels marked Before. The regions of the tissue section where carcinoma cells were removed by LCM are shown in the images marked After, and the isolated cells adhering to the CapSure LCM caps are shown in the images marked Capture. Each cap, containing 200–300 carcinoma cells, is extracted for RNA that is quantified and amplified before microarray analyses.

expression as well as the size and structure of RNA molecules requires retention of biological integrity. Because of the lability of mRNA, several workers have studied the effects of tissue fixation on RNA extraction and amplification after LCM,9,13 providing some insight into the stability of these labile molecules using current procedures. Advantages of LCM Manual microdissection techniques, which require tedious manipulation, significant manual dexterity, and a lengthy training program, are slow and the variability in tissue collection is significant. However, LCM, which uses standardized

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technology, allows rapid sample procurement of tissue structures with awkward geometry and efficient isolation of different cell types in close proximity or adjacent to each other.2–4,7 Furthermore, the transfer process is nondestructive and cell morphology is retained.7 Of particular importance in molecular diagnostics and gene discovery, there is a record of the original location of cells in the tissue and visual verification of cell capture. Some investigators have reported successful DNA analyses using 300–500 cells (e.g., Simone et al.,6 ; Sirivatanaukorn et al.14 ), while 500–1000 cells have been used to isolate RNA (e.g., Glasow et al.10 ; Luo et al.11 ; Goldsworthy et al.13 ). Examinations of proteins using a single technology have employed 1000– 5000 cells isolated by LCM (e.g., Banks et al.15 ; Emmert-Buck et al.16 ). Extraction and 2D PAGE of proteins from representative samples requires capturing 20,000–30,000 cells although new nanotechnology approaches are being developed.15,16 RNA Isolation, Characterization, and Amplification for Microarray In our laboratories, total RNA is isolated using the PicoPure (Arcturus) kits, which are optimized for extracting RNA from cells procured by LCM. Routinely 1–6 ng of total RNA may be isolated from 200–300 human breast cancer cells procured by LCM, using these reagents. The intactness of RNA in tissue sections is evaluated prior to proceeding with LCM by a variety of procedures including electrophoresis incorporating a series of markers of different base-pair lengths. For investigations of gene expression profiles of human tissues, we procure cells of interest (e.g., normal vs neoplastic) from at least three different regions of a single tissue section (Fig. 5). Note that the carcinoma cells were removed from each of the three regions of interest and procured cleanly and retained on the CapSure LCM caps (Fig. 5, lower images). Each cell capture (usually containing 200–1000 cells) is treated as an independent evaluation in that the RNA is extracted, purified, and amplified, then subjected to microarray (Fig. 6). RNA isolated from cells procured by LCM is amplified efficiently with the RiboAMP kits (Arcturus) enabling the production of microgram amounts of RNA from nanogram quantities isolated from breast carcinoma and normal cells. Amplification requires preparation of double-stranded cDNA from the mRNA fraction of total RNA followed by transcription in vitro. The use of exogenous primers maximizes reliability in the synthesis of cDNA template while reducing reaction 14

Y. Sirivatanaukorn, V. Sirivatanauksorn, S. Bhattacharya, B. R. Davidson, A. P. Dhillon, A. K. Kakkar, R. C. N. Williamson, and N. R. Lemoine, J. Pathol. 189, 344 (1999). 15 R. E. Banks, M. J. Dunn, M. A. Forbes, A. Stanley, D. Pappin, T. Naven, M. Gough, P. Harnden, and P. J. Selby, Electrophoresis 20, 689 (1999). 16 M. R. Emmert-Buck, J. W. Gillespie, C. P. Paweletz, D. K. Ornstein, V. Basrur, E. Appella, Q. H. Wang, J. Huang, N. Hu, P. Taylor, and E. F. Petricoin III, Mol. Carcinog. 27, 158 (2000).

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FIG. 6. A representative Eisen Color Map of the gene expression profiles of various human breast carcinomas. Although the microarray performed contained more than 12,000 genes, only a portion of the gene expression profile of each breast cancer is shown using the GeneMaths program (Applied Maths, Austin, TX). Note that without preconceived selection of criteria, gene clustering was observed. Through preliminary bioinformatic analyses, molecular signatures are being identified for several types of human breast cancers, such as those expressing estrogen receptors (ER+) compared with carcinomas lacking the receptor (ER−), which is a marker of anti-estrogen responsiveness. Principal component analysis (diagram on right) was performed using the data matrix shown on the left, and the collective results of the breast specimens are projected onto the three-dimensional space diagram using the first three components.

times. The aRNA prepared by this protocol is ready for labeling and hybridization necessary for microarray analyses. Preliminary studies of microarray analyses of independent amplifications from the same RNA preparation indicate an excellent correlation. RT-PCR was used to detect low-, medium-, and high-abundance genes within the amplified RNA population. Our laboratory has demonstrated that

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amplification of mRNA in all abundance classes ensures that differential gene expression patterns will be identified. Currently, we are using a microarray containing approximately 12,000 genes of which 10% were included through KnowledgeBased Selection based on reported alterations in cancer. From studies of more than 100 human breast cancers, we have demonstrated that the RNA isolated from LCM procured cells is intact for use in amplification of mRNA and subsequent microarray (Fig. 6). We are employing this approach to derive molecular signatures (gene expression profiles) to advance the classification of breast cancer and assessment of patient prognosis and therapeutic response. Additional Applications of Laser Capture Microdissection LCM is rapidly becoming the method of choice for selecting diseased cells from normal cells of the same tissue specimen for genomic and proteomic analyses. 7,10–16 Some of the applications of LCM in these areas related to molecular diagnostics and prognostics of human cancer are shown below. Genomics: Differential gene profiling Loss of heterozygosity Micro-satellite instability Gene quantification Mutation/clonal analysis Proteomics: Two-dimensional PAGE Western blots Immunoquantitation of proteins MALDI-TOF mass spectrometry The ability to procure homogeneous cell subpopulations of normal, premalignant, and malignant cell types and to accumulate data from each cell type advances our understanding of the underlying causes of tumor formation and permits the tracking, at the molecular level, of cell progression into a metastatic phenotype. Efforts are well underway to produce cDNA libraries that catalog genes differentially expressed during tumor progression (e.g., Peterson et al.17 ). The Cancer Genome Anatomy Project (CGAP) has utilized LCM to obtain normal and premalignant samples from human prostate, breast, ovary, colon, kidney, and 17

L. A. Peterson, M. R. Brown, A. J. Carlisle, E. C. Kohn, L. A. Liotta, M. R. Emmert-Buck, and D. B. Krizman, Cancer Res. 58, 5326 (1998).

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lung tissue, to name a few. Information from CGAP is publicly available through the CGAP-NIH Web site.18 Laser capture microdissection has proved to be a powerful tool for research into the cellular basis of disease and is increasingly being employed in drug discovery and clinical diagnostics. Physiological changes occurring during development and progression of normal cells to neoplastic lesions may be explored easily with LCM and proteomics and gene expression profiling. For clinical diagnosis, the ability to sample specific types of cells creates a new analytical paradigm which will allow patients to be diagnosed based on qualitative and quantitative gene expression as well as on levels of cell-specific proteins. As Wittliff suggested previously,1 a new generation of laboratory tests is rapidly evolving in which analyses will be performed directly on human tissue biopsies. It is envisioned that tissue banks such as the Biorepository at the Hormone Receptor Laboratory will be developed for long-term preservation of human tumor samples. This will allow assessment of genetic and biochemical properties of the stored tumor tissues as new clinical, chemical, and molecular biological probes are developed for cancer management, and as technologies such as laser capture microdissection are utilized to separate normal from tumor cells. 18

www.ncbi.nlm.nih.gov/CGAP

[3] Going in Vivo with Laser Microdissection ¨ By ANETTE MAYER, MONIKA STICH, DIETER BROCKSCH, KARIN SCHUTZE , and GEORGIA LAHR Introduction Tissue microdissection and single-cell isolation is one of the most advanced techniques in modern gene analysis and is especially useful for studying expression of genes in isolated tumor cells. Till now, microdissection methods have been limited to cells from fixed or frozen tissues.1–9 An old dream of cell biologists 1

W. Meier-Ruge, W. Bielser, E. Remy, F. Hillenkamp, R. Nitsche, and R. Unsöld, Histochem. J. 8, 387 (1976). 2 M. Schindler, M. L. Allen, M. R. Olinger, and J. F. Holland, Cytometry 6, 368 (1985). 3 Y. Kubo, F. Klimek, Y. Kikuchi, P. Bannasch, and O. Hino, Cancer Res. 55, 989 (1995). 4 M. R. Emmert-Buck, R. F. Bonner, P. D. Smith, R. F. Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, and L. A. Liotta, Science 274, 998 (1996). 5 R. F. Bonner, M. Emmert-Buck, K. Cole, T. Pohida, R. Chuaqui, S. Goldstein, and L. A. Liotta, Science 278, 1481 (1997).



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is to isolate living cells from tissue culture or unfixed and unfrozen sections of living tissues.10 But to date, laser-based microdissection of living cells resulted in the destruction of the isolated cells.2 Now, a modification of the laser microbeam microdissection (LMM) method in combination with the laser pressure catapulting (LPC) technique6 and a newly developed cell culture protocol allows microdissection and “ejection” of living single cells or cell clusters with ongoing cultivation for potential treatment and analysis. We established a unique technique in which cultured cells were microdissected and afterward catapulted by LPC into the cap of a microfuge tube. The viability of the catapulted cells is not affected as they enter the cell cycle and proliferate. Applying this protocol—select, microdissect, eject, and clone living cells—to biopsy slices will come true in the near future. As this, “going in vivo” opens up a broad spectrum of applications. Step I: Cell Culture Preparation A prerequisite for isolation of single living cells from cell cultures by laserassisted cell picking is the growth of the cells on a supporting membrane. The membrane is mounted in a specific cell culture chamber, the ROC chamber. For microdissection the membrane around the cell or cell clusters of interest is cut by the focused laser beam in a sufficient distance from the cell. Then the cell-membrane stack is catapulted by the laser beam into a conventional cap of a microfuge tube centered directly above the selected area (Fig. 1, A and B). Buffers, Reagents, and Equipment ROC chamber, Round Open Closed (PeCon and LaCon, Erbach-Bach, Germany) Polyethylene–naphthalene membrane, 1.35 µm (PEN membrane; P.A.L.M. Microlaser Technologies AG, Bernried, Germany) EJ28 cells, a bladder carcinoma cell line TPC-1 cells, a thyroid carcinoma cell line Dulbecco’s modified Eagle’s medium [Invitrogen GmbH (GIBCO-BRL), Karlsruhe, Germany] Dulbecco’s modified Eagle’s medium Nutrient Mixture F12-Ham (DME/F12 Hams) (Sigma-Aldrich GmbH, Deisenhofen, Germany) 200 mM L-glutamine (Sigma-Aldrich GmbH) 6

K. Schütze and G. Lahr, Nat. Biotechnol. 16, 737 (1998). K. Schütze, H. Pösl, and G. Lahr, Mol. Cell Biol. 44, 735 (1998). 8 G. Lahr, Lab. Invest. 80, 1 (2000). 9 G. Lahr, M. Stich, K. Sch¨ utze, P. Blümel, H. Pösl, and W. B. J. Nathrath, Pathobiology 68, 218 (2000). 10 M. Schindler, Nat. Biotechnol. 16, 719 (1998). 7

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FIG. 1. Schematic drawings of a cell culture grown on a PEN membrane in an ROC chamber. The chamber is attached to the microscope stage and the microfuge cap is centered above the line of laser fire directly in the ROC chamber (A). The selected cell-membrane stacks are microdissected by the laser beam (LMM). The cell-membrane stacks are catapulted by LPC directly into the cap of the sample tube supplied with a droplet of Hanks’ solution (B). The captured cells are covered with 25 µl of Hanks’ solution (C). The cap is topped with the remaining tube and the assembled tube is centrifuged to collect captured cells at the bottom of the microfuge tube (D).

10% Fetal calf serum (FCS; Sigma-Aldrich GmbH) 100× Antibiotic–antimycotic solution (Sigma-Aldrich GmbH) Hanks’ solution (Sigma-Aldrich GmbH) Trypsin–EDTA solution (Sigma-Aldrich GmbH) Conventional culture dish plates for cell culture Gassed incubator for cell culture Laser microscope, Robot-MicroBeam (P.A.L.M. Microlaser Technologies AG) Inverted microscope Axiovert 135 (Carl Zeiss, Göttingen, Germany) Microfuge tubes (P.A.L.M. Microlaser Technologies AG) Procedure Assembly of the ROC Chamber 1. Cover the glass bottom of the ROC chamber with the polyethylene– naphthalene membrane (PEN membrane) by using a droplet of 100% ethanol for mounting it onto the glass. 2. Expose the opened chamber with the membrane to UV light for 20 min to change the hydrophobic nature of the membrane into a more hydrophilic one. 3. Assemble the ROC chamber totally and autoclave it at 121◦ for 20 min.

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Cell Culture. The EJ28 bladder carcinoma cell line and the papillary thyroid tumor cell line TPC-1 (a generous gift of Dr. B. Mayr, Med. Hochschule Hannover, Germany) were used for the experiments. EJ28 cells were grown in Dulbecco’s modified Eagle’s medium and TPC-1 cells were grown in Dulbecco’s modified Eagle’s medium Nutrient Mixture F12-Ham (DME/F12 Hams), both supplemented with 5 mM L-glutamine, 10% fetal calf serum (FCS), and 1× antibiotic– antimycotic solution. Seed the cell culture cells at the desired density onto the membrane-covered ROC chamber in their appropriate medium. 4. Incubate the cells in the ROC chamber at 37◦ in a gassed incubator. After 1–2 days in culture the cells are ready for microdissection. Laser Microdissection and Catapulting 5. Remove the medium completely from the ROC chamber before laser microdissection (Figs. 1–4A). 6. Microdissect the desired cell-membrane sample. The parameters concerning laser energy and laser focus during microdissection (LMM) are dependent on the laser microscope system used and have to be optimized before use (Figs. 1A, 2B, 3B, 4C, 5B). 7. Apply a 10-µl droplet of Hanks’ solution on top of the selected cells to facilitate LPC. Be careful not to wash away the microdissected specimen.

FIG. 2. Images using LMM and LPC to capture 40 EJ28 cells. Cells before microdissection (A), after microdissection (B), cells remaining after LPC (C), catapulted membrane with the cells (D). 11 hr after plating (E), after 1 day (F), after 5 days (G), after 8 days (H), and after 12 days (I). Black arrow: cell filopodium. White arrow: mitotic cell. Dotted line: area to be microdissected. Bar equals 100 µm in A–D, F, and H; 50 µm in E, G, and I. Objective lenses in A–D and F: 20× ; E and I: 40× ; G: 5×; and H: 10×.

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FIG. 3. LMM and LPC images of a small TPC-1 cell cluster (10 cells). The sequence shows cells before LMM (A), after LMM (B), and remaining cell culture after LPC (C). (D) Aggregated cells within the “hanging droplet” 9 hr after collection. 1 day in culture the catapulted and aggregated cells begin to adhere to the bottom of the culture dish (E). Proliferating cells shown after 12 days in culture (F). Dotted line: area to be microdissected. Bar equals 50 µm in A–C; 100 µm in D–F. Objective lenses in A–C: 40×; D–F: 20×.

8. Pipette a 5-µl droplet of Hanks’ solution into the center of the cap of a microfuge tube and place the cap directly above the selected cells into the ROC chamber (Fig. 1A). 9. Catapult the cell-membrane stack with one single laser shot positioned at the border of the circumscribed membrane. For the catapulting the laser is focused below the microdissected target specimen. 10. Energy settings should be sufficiently high to catapult the microdissected cells with the membrane into a cap (Fig. 1, A and B). Even large cell-membrane stacks (for example 385× 248 µm) can be catapulted (Fig. 2C).

FIG. 4. Images of an experiment to destroy an “undesired cell” before LMM and LPC of about 18 living EJ28 cells. The sequence shows the cells before microdissection(A), after destruction of one specific cell (B), and after microdissection (C), the remaining cells after LPC (D), and the catapulted membrane with the cells (E). (F) Cells after 12 days in culture. Black arrow: cell destroyed by a precise laser shot. White arrow: cells on the membrane after catapulting. Dotted line: area to be microdissected. Bar equals 100 µm in A–E; 50 µm in F. Objective lenses in A–E: 20×; F: 10×.

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FIG. 5. Images of 5 pooled single EJ28 cells. The sequence shows the cells before LMM (A), after LMM of cell group 1 (B), after LMM of cell groups 1 and 2 (C), after LMM of cell groups 1 to 3 (D). The remaining cells after LPC of cell group 1 (E), 1 and 2 (F), and after LPC of cell group 3 (G). (H) 3 catapulted membranes with cells. Aggregated cells within the “hanging droplet” (I). Dotted lines: areas to be microdissected. Bar equals 100 µm in A–H; 50 µm in I. Objective lenses in A–H: 20×; I: 40×.

11. After LPC remove the ROC chamber from the microscope stage, take the cap, and inspect the catapulted cells in the cap now fixed within the manipulator (Figs. 2D, 4E, 5H). Notes to Step I a. To reduce the chance of contamination wear gloves during the whole cell culture procedure. Do not keep the cell cultures outside the incubator longer than necessary. In case of several experiments allow cells in the ROC chamber to recover from dryness by adding medium back to the cells. This medium has to be removed totally, otherwise during the cutting process the laser energy will be absorbed by the aqueous solution. This results in local heating of the medium and in visible steam bubbles, which destroy the viable cells. In addition, be aware that after several microdissection events the medium is entering the micro space between the membrane and the glass bottom of the ROC chamber. This makes further microdissection and catapulting more and more difficult and finally impossible. b. With the focused laser beam single cells or cell clusters are precisely separated together with the membrane from their surrounding (Figs 2B, 3B, 4C,

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5B–5D) using 20× (Figs. 2, 4, 5) or 40× (Fig. 3) objective lenses. Laser circumscription of the cell-membrane stack results in a gap, free of any other biological material, separating the target from its surroundings.6 The width of the gap is about 3–5 µm, depending on the objective lens and the absorption behavior of the specimen. Where the selected specimen area contains an undesired cell, this cell can be eliminated by a direct laser shot (Fig. 4B). c. Increased laser energy catapults the target specimen into the cap of a microfuge tube. Even large cell-membrane stacks (for example 290× 369 µm) can be catapulted (Figs. 2D, 4E, 5H). This results in empty patches within the cell culture (Figs. 2C, 3C, 4D, 5G). The laser-catapulted cell-membrane stacks are well preserved and allow direct correlation with their templates in terms of shape, size, and original position (Figs. 2D, 4E, 5H). Microdissection and catapulting of cell clusters in these large sizes takes less than 2 min. The manipulation of single living cells is done within seconds. Laser-assisted isolation is performed with cell clusters of about 10 cells (Fig. 3) and several tens (Figs. 2 and 4), as well as single cells (Fig. 5).

Step II: Collection of Catapulted Cells Procedure 1. Cover the catapulted cells in the cap with 25 µl of Hanks’ solution. 2. Close the cap with the remaining tube and store for up to 30 min at room temperature. 3. Centrifuge the tube for 1 min at 8000g and discard the supernatant. 4. Resuspend the cells in 20 µl trypsin–EDTA solution and incubate for 10 min at room temperature to detach the cells from the membrane. 5. Centrifuge for 1 min at 8000g. 6. After centrifugation the trypsinized cells (pellet) are resuspended in 15 µl (

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