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Apoptosis Resource
Beginning of the End
Induction of Apoptosis
Changes at the Plasma Membrane
Changes in the Mitochondria
Changes in the Cytoplasm
Changes in the Nucleus
Measurement of Cell Proliferation
Appendix
   
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Apoptosis Resource: Changes in the Cytoplasm
 
Apoptosis: The Beginning of the End
Induction of Apoptosis
Measurement of Apoptosis-Induced Changes at the Plasma Membrane
Measurement of Apoptosis-Induced Changes in the Mitochondria
Measurement of Apoptosis-Induced Changes in the Cytoplasm
Measurement of Apoptosis-Induced Changes in the Nucleus
Measurement of Cell Proliferation
Appendix
 Technical Tips
Measurement of Apoptosis-Induced Changes in the Cytoplasm
Activation of caspases is one of the most widely recognized features of apoptosis. Caspases are cysteine-dependent, aspartate-specific proteases. They exist as latent precursors in the cytoplasm which, upon activation, destroy key components of the cellular infrastructure. Thus far fourteen members of the caspase family have been identified, eleven of these are present in humans. Caspases can be subdivided into three groups. Upstream initiators include caspase-2, -8, -9, and -10. Downstream executioners include caspase-3, -6, and -7. A third group includes members involved in the inflammatory process are caspase-1, -4, -5, and -12. A distinctive feature of caspases is the absolute requirement of an aspartic acid residue in the substrate P1 position. The P4 residue is important in substrate recognition and specificity. Generally, catalysis involves a cysteine protease mechanism. Measurement of caspase activity is based on a tetrapeptide corresponding to substrate P4-P1 residues coupled either to a colorimetric (pNA) or fluorogenic (AFC, AMC) compound. Upon caspase-mediated peptide cleavage, the free colorimetric or fluorogenic group is released and can be measured by spectrophotometric or fluorometric methods. This procedure is useful for measurement of caspase activity in cell lysates.

Reference:
S . J . Riedel and Y . Shi . 2004 . Nat. Rev. Molecular Cell Biol. 5,897 .
 
 
Measurement of Changes in the Cytoplasm: Fluorescent-Based Detection Kits
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Caspase-3 Intracellular Activity Assay Kit I (PhiPhiLux® G1D2) Fluorescence Intact cells 30 Tests 235430
Caspase-3 Intracellular Activity Assay Kit II (PhiPhiLux® G2D2) Fluorescence Intact cells 30 Tests 235432
Note: FC: flow cytometry; IF: immunofluorescence
 
Measurement of Changes in the Cytoplasm: Substrate-Based Caspase Detection Kits
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Caspase-8 Assay Kit Colorimetric or Fluorimetric Purified protein or induced cell lysates 96 Tests 218770
Caspase-1 Assay Kit, Colorimetric Colorimetric Cell lysates 100 Tests 218790
Caspase-1 Assay Kit, Fluorometric Fluorometric cell lysates 100 Tests 218791
Caspase-1 Inhibitor Screening Assay Kit Colorimetric Purified protein 96 Tests 218734
Caspase-2 Assay Kit, Colorimetric Colorimetric Cell lysates 100 Tests 218792
Caspase-2 Assay Kit, Fluorometric Fluorometric Cell lysates 96 Tests 218793
Caspase-3 Activity Assay Kit, Fluorometric Fluorometric Cell lysates 96 Tests QIA70
Caspase-3 Activity Assay Kit, HTS Fluorometric Cultured cells or purified protein 100 or 500 Tests HTS02
Caspase-3 Cellular Activity Assay Kit Fluorometric or colorimetric Cell extracts 96 Tests 235419
Caspase-3 Immunoassay/Activity Kit, Fluorometric Fluorometric Cultured cells 96 Tests QIA107
Caspase-3 Inhibitor Screening Assay Kit Fluorometric or Colorimetric Recombinant or purified caspase-3 96 Tests 235418
Caspase-5 Assay Kit, Colorimetric Colorimetric Cell lysate 100 Tests 218804
Caspase-6 Assay Kit, Colorimetric Colorimetric Cell lysate 100 Tests 218802
Caspase-7 Immunoassay/Activity Kit, Fluorometric Fluorometric Cultured cells 96 Tests QIA108
Caspase-8 Activity Assay Kit, Fluorometric Fluorometric Cell lysate 96 Tests QIA71
Active Caspase-8 Assay Kit Fluorometric Cells 182 Tests CBA046
Caspase-9 Activity Assay Kit, Fluorometric Fluorescence Cell lysate 96 Tests QIA72
Caspase-9 Activity Assay, HTS Fluorescence Purified protein, cell lysate, or cultured cells 96 Tests HTS04
Active Caspase-9 Assay Kit Fluorometric Cells 480 Tests CBA047
Caspase-9 Assay Kit, Colorimetric Colorimetric Cell lysate 100 Tests 218824
Caspase-10 Assay Kit, Fluorometric Fluorometric Cell lysates 96 Tests 218811
 
Detection of active Caspase-9 in lysates from HepG2 hepatocytes, G361 melanoma cells and Jurkat T-cells. Caspase-9 was activated by incubating the indicated amount of cell lysates with cytochrome c and dATP. The data show that detection of Caspase- 9 is proportional to the amount of activated cell extracts assayed.
Active Caspase-9 Assay Kit Cat. No. CBA047
The Active Caspase-9 Assay Kit is useful for studying the effects of biochemical compounds on caspase-9 activity in cell lysates. The assay employs a monoclonal antibody specific for caspase-9 coated onto the wells of a 96-well plate. The immunocapture of active caspase-9 is followed by the detection of caspase-9 activity by cleavage of a fluorogenic substrate. Fluorescence is measured in a 96- well fluorescent plate reader.
 
 
Use of Fluorochrome - Inhibitor Conjugates for Active Caspase Detection
Caspase inhibitors act by binding to the active site of the caspase, either in a reversible or in an irreversible manner. Inhibitor design includes a peptide recognition sequence attached to a functional group such as an aldehyde (CHO), chloromethylketone (CMK), or fluoromethylketone (FMK). The peptide recognition sequence corresponding to that found in endogenous substrates determines the specificity of a particular caspase. Compounds with the Ac-YVADCHO sequence are potent inhibitors of caspase-1 (Ki~10 nM), and exhibit very weak inhibitory effect on capases-3 and 7 (Ki > 50 uM). Exclusion of the amino acid from the P4 position of the inhibitor peptide results in a potent, but less specific inhibitor like Z-VAD-FMK, which acts as a pan caspase inhibitor, and inhibits most caspases. The inhibitor can also be tagged with a fluorochrome, such as FITC, that can be used as a detection tool for activated caspases. For example, FITC conjugated to VAD-FMK provides a very useful, cell permeable, non-toxic inhibitor that binds irreversibly to activated caspases in apoptotic cells. The fluorescence intensity can be measured by fluorescence microscopy, fluorescence plate reader, or flow cytometry.
 
Measurement of Changes in the Cytoplasm:
Fluorochrome - Inhibitor Conjugates for Active Caspase Detection
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Caspase Detection Kit (FITC-VAD-FMK) Fluorescence Intact cells 100 Tests QIA90
Caspase Detection Kit (Red-VAD-FMK) Fluorescence Intact cells 100 Tests QIA92
Caspase-3 Detection Kit (FITC-DEVD-FMK) Fluorescence Cultured cells 100 Tests QIA91
Caspase-3 Detection Kit (Red-DEVD-FMK) Fluorescence Cultured cells 100 Tests QIA93
Active Caspase-3 ELISA Kit Colorimetric Cells 96 Tests CBA045
Caspase-8 Detection Kit (FITC-IETD-FMK) Fluorescence Intact cells 100 Tests QIA113
Caspase-8 Detection Kit (Red-IETD-FMK) Fluorescence Intact cells 100 Tests QIA114
Caspase-9 Detection Kit (FITC-LEHD-FMK) Fluorescence Intact cells 100 Tests QIA115
Caspase-9 Detection Kit (Red-LEHD-FMK) Fluorescence Intact cells 100 Tests QIA116
Note: ELISA: enzyme linked immunosorbent assay; FC: flow cytometry; IF:immunofluorescence
 
 
Use of Immunoblot - Based Caspase Detection Kits
Caspases destroy key components of the cellular infrastructure and activating factors that mediate damage to the cells. Over 280 caspase targets have been identified to date. Proteins cleaved by these caspases are involved in cell cycle progression/regulation, cellular repair, cytoskeletal architecture and structure, DNA synthesis, cleavage, and repair, as well as cell detachment and cytokine precursors. The majority of substrates identified are cleaved by caspase-3 and some of these same substrates can also be cleaved by caspase-7. The demonstration of caspase substrate cleavage is an indirect way to measure caspase activity during apoptosis. Thus, analysis of changes in the molecular weight of caspase substrates can be measured by SDS-PAGE followed by immunoblot detection with specific substrate antibodies. Antibodies are available that are specific for the procaspase or activated form of certain caspases. Some of the antibodies recognize both the uncleaved (Pro) and cleaved (active) forms of the caspase. This procedure has made it possible to detect endogenous activated caspases or the resultant apoptosis-induced proteolysis of substrates.

Reference:
Fischer, U . et al . 2003 . Cell Death and Differentiation 10, 76 .
 
Measurement of Changes in the Cytoplasm: Immunoblot Kits
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Cleaved Caspase Antibody Sampler Kit IB AP1026
Downstream Effector Caspase Sampler Kit IB ASK23
Note: IB: immunoblotting
 
Cleaved Caspase Antibody Sampler Kit
Cat. No. AP1026
Detection of cleaved caspase-3 by immunoblotting . Samples: Cell lysates from HeLa, NIH/3T3, and C6 cells, untreated, staurosporine-treated (1 mM in vitro) or cytochrome c-treated (0 .25 mg/ml in vitro) . Primary antibody: Anti-Cleaved Caspase-3 (Asp175) (Cat. No. AP1027) (1:1000) . Detection: chemiluminescence . Representative data from Cat. No. AP1026
 
 
 
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