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  • Life, Death, and Cell ProliferationA product guide to Apoptosis, Cell Cycle and Cytotoxicity

  • Table of Contents

    Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

    Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

    Plasma Membrane Alterations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

    Annexin V Reagents and Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

    Mitochondrial Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

    Mitochondrial Membrane Potential - m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6Activation of Caspases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

    Active Caspase-3 and Other Caspases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

    BD Cytometric Bead Array (CBA) Multiplexed Quantitation/Analysis of Caspase-3, PARP, Bcl-2 . . . . . . . . . . . . . . . . .9

    Active Caspase-3 ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10

    Caspase Activity Assays: Kits and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

    QTL Lightspeed Enzyme Activity Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13

    Membrane Permeable Caspase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

    Analysis of Caspase Cleavage Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16

    DNA Fragmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18

    Individual Antibodies to Proteins Involved in Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19

    Bcl-2 Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19

    Signaling Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20

    BD PowerBlotSM: Protein Array Screening Service . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25

    Summary. Application Decision Tree. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26

    Cell Cycle and Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27

    Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27

    Analysis of Cellular DNA Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28Cell Proliferation Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30

    BrdU Flow Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30

    Phosphorylation of Serine 10 in Histone H3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31

    BrdU In Situ Detection Kit, IHC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32

    Analysis of Cyclins, Cyclin Dependent Kinases, and Cell Cycle Regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33

    Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36

    ADME/TOX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36Genotoxicicology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38

    DNA Damage Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38

    icroFlow Micronucleus Analysis Kits. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40Immunotoxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41

    Cellular Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43

    BD Pathway Live Cell Confocal Bioimager (New) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43BD Flow Cytometry Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .44RNA. Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46

    BD RiboQuant Multi-Probe Ribonuclease Protection Assay (RPA) System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46

    2

    Reactivity: Bab:baboon; Bov:Bovine; C: Chicken; Cyno: Cynomolgus; D: Dog; Dros: Drosophila; F: Frog; GPig: Guinea Pig; Gt: Goat; Ham: Hamster; Hs: Horse; Hu: Human; Koa: Koala; Mam:Mammalian; Ms: Mouse; Pg: Pigeon; Qua: Quail; R: Rat; Rb: Rabbit; Rhe: Rhesus; Tr: Trout; Xen: Xenopus

    Applications:Block: Blocking; FA: Functional Assay; FCM: Flow Cytometry; IC/FCM: Intracellular Flow Cytometry; IF: Immunofluorescence; IHC: Immunohistochemistry; IP: Immunoprecipitation;Neu: Neutralizing; RPA: Ribonuclease Protection Assay; WB: Western Blot

    For Research Use Only. Not for use in diagnostic or therapeutic procedures.All applications are either tested in-house or reported in the literature. See Technical Data Sheets for details.2005 Becton, Dickinson and Company. All rights reserved. No part of this publication may be reproduced, transmitted, transcribed, stored in retrieval systems,or translated into any language or computer language, in any form or by any means: electronic, mechanical, magnetic, optical, chemical, manual, or otherwise, withoutprior written permission from BD Biosciences.BD flow cytometers are class I (1) laser products.The Alexa Fluor dye and Cascade Blue dye antibody conjugates in this product are sold under license from Molecular Probes, Inc., for research use only or as analyte specificreagents, except for use in combination with microarrays and high content screening, and are covered by pending and issued patents.BD, BD Logo and all other trademarks are the property of Becton, Dickinson and Company. 2005 BD

    www.bdbiosciences.com

  • Introduction

    Programmed cell death or Apoptosis is anormal physiological process that occursduring embryonic development and in themaintenance of tissue homeostasis. Theterm apoptosis, from the Greek word forfalling off of leaves from a tree, is usedto describe a process in which a cell activelyparticipates in its own destructive processes.The apoptotic program is characterized bycertain morphological features. Theseinclude changes in the plasma membranesuch as loss of membrane symmetry andloss of membrane attachment, acondensation of the cytoplasm andnucleus, and internucleosomal cleavageof DNA. In the final stages, the dying cellsbecome fragmented into apoptoticbodies, which are rapidly eliminated byphagocytic cells without eliciting significantinflammatory damage to surrounding cells.Inappropriate induction of apoptosis haswidespread pathological implications andhas been associated with diseases such asneurodegenerative diseases, graft-versus-host diseases, transplant rejection, cancer,and AIDS.

    The study of apoptosis has never beenmore important than it is today. Alongwith this intensive study has come thedevelopment of a multitude of methods to d

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