Apoptosis and Cell Proliferation

BOEHRINGER MANNHEIMApoptosis and Cell Proliferation2nd editionIntended UseOur preparations are exclusively intended for analytical purposes or for studies based on animal experiments. They must notbe used for human beings since they were neither tested nor intended for such utilization.Unsere Präparate sind ausschließlich für analytische Zwecke oder tierexperimentelle Studien bestimmt. Sie dürfen am Menschen nicht angewandt werden, weil sie hierfür weder geprüft noch vorgesehen sind.Nuestros preparados están destinados exclusivamente a fines analíticos o estudios experimentales con animales. No debenser administrados o aplicados a seres humanos por no estar previstos a tal efecto y no haber sido sometidos a la verificacióncorrespondiente.Nos préparations sont exclusivement réservées soit à des fins analytiques, soit à des études basant sur des expériences avecdes animaux.

Elle ne doivent en aucun cas êntre utilisées sur l’être humain car elles ne sont ni vérifiées, ni prévues à ces fins.AcknowledgementWe would like to thank all contributors and editors for their diligent efforts. Without their work, this project would not havebeen possible. Finally, we are especially honored and delighted that Dr. Andrew Wyllie agreed to write the introduction tothe Cell Death chapter.Contributors:Andrew Wyllie, Ph.DVicki Donahue, M.S.Bertram Fischer, Ph.D.David Hill, Ph.D.Joe Keesey, Ph.D.Simone Manzow, Ph.D.Editorial Management:Doris EiselGeorg Fertig, Ph.D.Bertram Fischer, Ph.D.Simone Manzow, Ph.D.Karl SchmeligArt Direction andTypesetting:typoPlus Föll + Schulz GmbHMannheimCover:“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, BiochemicaAll rights reserved. No part of this booklet may be reproduced in any form without written permission of the publishers.Printed in GermanyApoptosis and Cell Proliferation2nd editionIChapter 1Cell Death – Apoptosis and Necrosis1.1Introduction ___________________________________________________________ 21.1.11.1.21.1.3Terminology of cell death ________________________________________________________________ 2Differences between necrosis and apoptosis _________________________________________________ 3Apoptotic Pathways ____________________________________________________________________ 41.2Apoptosis Assay Methods _________________________________________________ 61.2.11.2.1.11.2.1.21.2.1.31.2.21.2.2.11.2.2.21.2.2.31.2.2.41.2.31.3P Method/Product selection guide __________________________________________________________ 7Methods for studying apoptosis in cell populations ____________________________________________ 8Assays that measure DNA fragmentation ______________________________________________________ 8P Apoptotic DNA Ladder Kit ______________________________________________________________ 11P Cell Death Detection ELISAPLUS __________________________________________________________ 13Assays that measure apoptosis-induced proteases (caspases) _____________________________________ 16P Caspase 3 Activity Assay_______________________________________________________________ 17P Anti-PARP _________________________________________________________________________ 20Summary of methods for studying apoptosis in cell populations ____________________________________ 22Methods for studying apoptosis in individual cells____________________________________________ 24The TUNEL enzymatic labeling assay ________________________________________________________ 24P In Situ Cell Death Detection Kit, Fluorescein _________________________________________________ 27P In Situ Cell Death Detection Kit, AP _______________________________________________________ 29P In Situ Cell Death Detection Kit, POD ______________________________________________________ 29Assays that measure membrane alterations ___________________________________________________ 31P Annexin-V-FLOUS____________________________________________________________________ 32P Annexin-V-FLOUS Staining Kit ___________________________________________________________ 32P Annexin-V-Alexa™ 568 _______________________________________________________________ 32P Annexin-V-Biotin ____________________________________________________________________ 34Assays that use DNA stains _______________________________________________________________ 36P DAPI, Ethidium bromide, Propidium iodide __________________________________________________ 36Summary of methods for studying apoptosis in individual cells _____________________________________ 38Detection of apoptosis-related proteins ____________________________________________________ 40P Anti-Fas (CD95/Apo-1) ________________________________________________________________ 41P Anti-Fas-Biotin (CD95/Apo-1) ___________________________________________________________ 42P Anti-Bcl-2 oncoprotein, human __________________________________________________________ 44P Anti-p53-Protein, mutant ______________________________________________________________ 46P Anti-p53-Protein pan _________________________________________________________________ 46P Anti-p53 pan _______________________________________________________________________ 46P Anti-p53, Biotin labeled _______________________________________________________________ 46P Anti-p53, Peroxidase labeled____________________________________________________________ 46P p53 pan ELISA ______________________________________________________________________ 48Cytotoxicity Assay Methods_______________________________________________501.3.1 Relationship between cytotoxicity, apoptosis and necrosis _____________________________________ 501.3.2 Methods for studying cytotoxicity ________________________________________________________ 501.3.2.1 Assays that measure plasma membrane leakage _______________________________________________ 51P Cytotoxicity Detection Kit (LDH) __________________________________________________________ 52P Cellular DNA Fragmentation ELISA________________________________________________________ 541.3.2.2 Assays that measure metabolic activity ______________________________________________________ 58P Cell Proliferation Kit I (MTT) _____________________________________________________________ 58P Cell Proliferation Kit II (XTT) _____________________________________________________________ 58P Cell Proliferation Reagent WST-1_________________________________________________________ 581.3.2.3 Summary of methods for studying cytotoxicity _________________________________________________ 60IIChapter 2Cell Proliferation and Viability2.1Introduction ___________________________________________________________ 642.1.12.1.2Terminology of cell proliferation and viability _______________________________________________ 64Cell cycle ___________________________________________________________________________ 642.2Cell proliferation/viability assay methods _____________________________________ 67P Method/Product selection guide _________________________________________________________ 682.2.1 Methods for studying cell proliferation and viability in cell populations ___________________________ 702.2.1.1 Assays that measure metabolic activity ______________________________________________________ 70P Cell Proliferation Kit I (MTT) _____________________________________________________________ 73P Cell Proliferation Kit II (XTT) _____________________________________________________________ 74P Cell Proliferation Reagent WST-1_________________________________________________________ 752.2.1.2 Assays that measure DNA synthesis ________________________________________________________ 77P BrdU Labeling and Detection Kit III________________________________________________________ 79P Cell Proliferation ELISA, BrdU (colorimetric) _________________________________________________ 81P Cell Proliferation ELISA, BrdU (chemiluminescence) ___________________________________________ 812.2.1.3 Summary of methods for studying cell proliferation and cell viability in cell populations ___________________ 842.2.1.4 Single reagents for the measurement of DNA synthesis___________________________________________ 842.2.2 Methods for studying cell proliferation and viability in individual cells ____________________________ 862.2.2.1 Assays that measure DNA synthesis ________________________________________________________ 86P BrdU Labeling and Detection Kit I_________________________________________________________ 87P BrdU Labeling and Detection Kit II ________________________________________________________ 87P In Situ Cell Proliferation Kit, FLUOS _______________________________________________________ 88P In Situ Cell Proliferation Kit, AP __________________________________________________________ 90P Anti-BrdU, formalin grade ______________________________________________________________ 93P Anti-BrdU-Fluorescein_________________________________________________________________ 93P Anti-BrdU-AP, F(ab’)2 fragments _________________________________________________________ 93P Anti-BrdU-Peroxidase, Fab fragment ______________________________________________________ 932.2.2.2 Assays that monitor expression of cell cycle-associated antigens ___________________________________ 96P Monoclonal antibodies to cell cycle-associated antigens________________________________________ 972.2.2.3 Summary of methods for studying cell proliferation and viability in individual cells ______________________ 100IIIChapter 3Appendix3.1Technical tips _________________________________________________________1043.1.13.1.23.1.2.13.1.2.23.1.2.33.1.33.1.43.1.5Selected frequently asked questions (FAQs) about cell death assays ____________________________Technical tips on the TUNEL method _____________________________________________________TUNEL: Improvement and evaluation of the method for in situ apoptotic cell identification_________________TUNEL protocol for tissues which tend to give false positives______________________________________Tips for avoiding or eliminating potential TUNEL labeling artifacts __________________________________Technical tips on the use of Annexin-V-Biotin for light microscope detection______________________Technical tips on the use of the Apoptotic DNA Ladder Kit on tissue samples______________________Technical tips on the Cell Proliferation ELISA kits ___________________________________________3.2Special applications of cell death and cell proliferation methods___________________1111041051051051071091091103.2.1 TUNEL assays _______________________________________________________________________ 1113.2.1.1 Discrimination between dead and viable apoptotic cells using two-color TdT assay and surface labeling asdetected by flow cytometry ______________________________________________________________ 1113.2.1.2 The use of flow cytometry for concomitant detection of apoptosis and cell cycle analysis _________________ 1113.2.1.3 Comparison of two cell death detection methods: In situ nick translation and TUNEL ____________________ 1123.2.1.4 Fixation of tissue sections for TUNEL combined with staining for thymic epithelial cell marker______________ 1123.2.2 Metabolic assays ____________________________________________________________________ 1133.2.2.1 Biochemical and cellular basis of cell proliferation assays that use tetrazolium salts_____________________ 1133.2.3 Annexin assays______________________________________________________________________ 1133.2.3.1 The use of annexin for concomitant detection of apoptosis and cellular phenotype ______________________ 1133.2.4 BrdU assays ________________________________________________________________________ 1143.2.4.1 Detection of bromodeoxyuridine in paraffin-embedded tissue sections using microwave antigen retrieval isdependent on the mode of tissue fixation ____________________________________________________ 1143.3References ___________________________________________________________1153.3.13.3.23.3.3Apoptosis-related parameters – Abbreviations and References_________________________________ 115Examples for applications of Boehringer Mannheim products __________________________________ 120General references ___________________________________________________________________ 1273.4General abbreviations ___________________________________________________1293.5Ordering Guide ________________________________________________________1313.6Index _______________________________________________________________134IVOverview of this GuideHow this guide can help you study cell death and cell proliferation?When and why do cells die? Does the concentration of environmental pollutants exert cytotoxic or cytostatic effects on cells?What factors influence the rate and timing of cell proliferation? Researchers in basic, industrial, and medical research are asking these questions and looking for answers.