Apoptosis and Cell Proliferation

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Transcript of Apoptosis and Cell Proliferation

BOEHRINGER MANNHEIM

Apoptosis and Cell Proliferation2nd edition

Intended UseOur preparations are exclusively intended for analytical purposes or for studies based on animal experiments. They must not be used for human beings since they were neither tested nor intended for such utilization. Unsere Prparate sind ausschlielich fr analytische Zwecke oder tierexperimentelle Studien bestimmt. Sie drfen am Menschen nicht angewandt werden, weil sie hierfr weder geprft noch vorgesehen sind. Nuestros preparados estn destinados exclusivamente a fines analticos o estudios experimentales con animales. No deben ser administrados o aplicados a seres humanos por no estar previstos a tal efecto y no haber sido sometidos a la verificacin correspondiente. Nos prparations sont exclusivement rserves soit des fins analytiques, soit des tudes basant sur des expriences avec des animaux. Elle ne doivent en aucun cas ntre utilises sur ltre humain car elles ne sont ni vrifies, ni prvues ces fins.

AcknowledgementWe would like to thank all contributors and editors for their diligent efforts. Without their work, this project would not have been possible. Finally, we are especially honored and delighted that Dr. Andrew Wyllie agreed to write the introduction to the Cell Death chapter.

Contributors:

Andrew Wyllie, Ph.D Vicki Donahue, M.S. Bertram Fischer, Ph.D. David Hill, Ph.D. Joe Keesey, Ph.D. Simone Manzow, Ph.D.

Editorial Management:

Doris Eisel Georg Fertig, Ph.D. Bertram Fischer, Ph.D. Simone Manzow, Ph.D. Karl Schmelig

Art Direction and Typesetting:

typoPlus Fll + Schulz GmbH Mannheim

Cover: Dispholidus typus This is the original picture of the snake on the cover. The colors were changed for design reasons only.

1998 by Boehringer Mannheim GmbH, Biochemica All rights reserved. No part of this booklet may be reproduced in any form without written permission of the publishers. Printed in Germany

Apoptosis and Cell Proliferation2nd edition

I

Chapter 1 Cell Death Apoptosis and Necrosis1.11.1.1 1.1.2 1.1.3

Introduction ___________________________________________________________ 2Terminology of cell death ________________________________________________________________ 2 Differences between necrosis and apoptosis _________________________________________________ 3 Apoptotic Pathways ____________________________________________________________________ 4

1.21.2.1 1.2.1.1

Apoptosis Assay Methods _________________________________________________ 6b Method/Product selection guide __________________________________________________________ 7 Methods for studying apoptosis in cell populations ____________________________________________ 8 Assays that measure DNA fragmentation ______________________________________________________ 8 b Apoptotic DNA Ladder Kit ______________________________________________________________ 11 b Cell Death Detection ELISAPLUS __________________________________________________________ 13 Assays that measure apoptosis-induced proteases (caspases) _____________________________________ 16 b Caspase 3 Activity Assay_______________________________________________________________ 17 b Anti-PARP _________________________________________________________________________ 20 Summary of methods for studying apoptosis in cell populations ____________________________________ 22 Methods for studying apoptosis in individual cells____________________________________________ 24 The TUNEL enzymatic labeling assay ________________________________________________________ 24 b In Situ Cell Death Detection Kit, Fluorescein _________________________________________________ 27 b In Situ Cell Death Detection Kit, AP _______________________________________________________ 29 b In Situ Cell Death Detection Kit, POD ______________________________________________________ 29 Assays that measure membrane alterations ___________________________________________________ 31 b Annexin-V-FLOUS____________________________________________________________________ 32 b Annexin-V-FLOUS Staining Kit ___________________________________________________________ 32 b Annexin-V-Alexa 568 _______________________________________________________________ 32 b Annexin-V-Biotin ____________________________________________________________________ 34 Assays that use DNA stains _______________________________________________________________ 36 b DAPI, Ethidium bromide, Propidium iodide __________________________________________________ 36 Summary of methods for studying apoptosis in individual cells _____________________________________ 38 Detection of apoptosis-related proteins ____________________________________________________ 40 b Anti-Fas (CD95/Apo-1) ________________________________________________________________ 41 b Anti-Fas-Biotin (CD95/Apo-1) ___________________________________________________________ 42 b Anti-Bcl-2 oncoprotein, human __________________________________________________________ 44 b Anti-p53-Protein, mutant ______________________________________________________________ 46 b Anti-p53-Protein pan _________________________________________________________________ 46 b Anti-p53 pan _______________________________________________________________________ 46 b Anti-p53, Biotin labeled _______________________________________________________________ 46 b Anti-p53, Peroxidase labeled____________________________________________________________ 46 b p53 pan ELISA ______________________________________________________________________ 48

1.2.1.2

1.2.1.3 1.2.2 1.2.2.1

1.2.2.2

1.2.2.3 1.2.2.4 1.2.3

1.3

Cytotoxicity Assay Methods_______________________________________________50

1.3.1 Relationship between cytotoxicity, apoptosis and necrosis _____________________________________ 50 1.3.2 Methods for studying cytotoxicity ________________________________________________________ 50 1.3.2.1 Assays that measure plasma membrane leakage _______________________________________________ 51 b Cytotoxicity Detection Kit (LDH) __________________________________________________________ 52 b Cellular DNA Fragmentation ELISA________________________________________________________ 54 1.3.2.2 Assays that measure metabolic activity ______________________________________________________ 58 b Cell Proliferation Kit I (MTT) _____________________________________________________________ 58 b Cell Proliferation Kit II (XTT) _____________________________________________________________ 58 b Cell Proliferation Reagent WST-1_________________________________________________________ 58 1.3.2.3 Summary of methods for studying cytotoxicity _________________________________________________ 60 II

Chapter 2 Cell Proliferation and Viability2.12.1.1 2.1.2

Introduction ___________________________________________________________ 64Terminology of cell proliferation and viability _______________________________________________ 64 Cell cycle ___________________________________________________________________________ 64

2.2

Cell proliferation/viability assay methods _____________________________________ 67

b Method/Product selection guide _________________________________________________________ 68 2.2.1 Methods for studying cell proliferation and viability in cell populations ___________________________ 70 2.2.1.1 Assays that measure metabolic activity ______________________________________________________ 70 b Cell Proliferation Kit I (MTT) _____________________________________________________________ 73 b Cell Proliferation Kit II (XTT) _____________________________________________________________ 74 b Cell Proliferation Reagent WST-1_________________________________________________________ 75 2.2.1.2 Assays that measure DNA synthesis ________________________________________________________ 77 b BrdU Labeling and Detection Kit III________________________________________________________ 79 b Cell Proliferation ELISA, BrdU (colorimetric) _________________________________________________ 81 b Cell Proliferation ELISA, BrdU (chemiluminescence) ___________________________________________ 81 2.2.1.3 Summary of methods for studying cell proliferation and cell viability in cell populations ___________________ 84 2.2.1.4 Single reagents for the measurement of DNA synthesis___________________________________________ 84 2.2.2 Methods for studying cell proliferation and viability in individual cells ____________________________ 86 2.2.2.1 Assays that measure DNA synthesis ________________________________________________________ 86 b BrdU Labeling and Detection Kit I_________________________________________________________ 87 b BrdU Labeling and Detection Kit II ________________________________________________________ 87 b In Situ Cell Proliferation Kit, FLUOS _______________________________________________________ 88 b In Situ Cell Proliferation Kit, AP __________________________________________________________ 90 b Anti-BrdU, formalin grade ______________________________________________________________ 93 b Anti-BrdU-Fluorescein_________________________________________________________________ 93 b Anti-BrdU-AP, F(ab)2 fragments _________________________________________________________ 93 b Anti-BrdU-Peroxidase, Fab fragment ______________________________________________________ 93 2.2.2.2 Assays that monitor expression of cell cycle-associated antigens ___________________________________ 96 b Monoclonal antibodies to cell cycle-associated antigens________________________________________ 97 2.2.2.3 Summary of methods for studying cell proliferation and viability in individual cells ______________________ 100

III

Chapter 3 Appendix3.13.1.1 3.1.2 3.1.2.1 3.1.2.2 3.1.2.3 3.1.3 3.1.4 3.1.5

Technical tips _________________________________________________________