Apoptosis, defined as programmed cell death, plays a very important role in many physiological and pathological conditions such as embryo and organ development, immune responses, tumor development and growth. Detecting apoptotic cells or monitoring the cells progressing to apoptosis is an essential step in basic research and in developing drugs that may regulate apoptosis.

Apoptosis is characterized by many biological and morphological changes; such as, change of mitochondrial membrane potential, activation of caspases, DNA fragmentation, membrane blebbing and formation of apoptotic bodies. Based on these changes, various assays are designed to detect or quantitate apoptotic cells. Typical assays include Annexin- V binding, caspase enzyme activity, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling) and DNA gel electrophoresis (see Table 1, next page).

The goal of this manual is to provide customers with proven methods that highlight the menu of products offered by BioSource International, Inc. Our Research and Development staff has detailed the protocols used to obtain the results given here. When appropriate, buffer formulations and specific parameters for instruments are also provided. As always, your feedback has contributed to the initiative for this publication and will continue to affect improvements in the materials which are supplied with our products. Please contact our Technical Service staff with any inquiries regarding this manual. (tech.support@biosource.com or 1-800-242-0607.)

The first method which is described in this publication is the detection of Annexin V-FITC by flow cytometry. This method has become a hallmark for the separation of necrotic cells from those undergoing true apoptosis. BioSource International, Inc. has formulated a kit containing Annexin V-FITC and propidium iodide, along with detailed instructions on how to distinguish cells in early apoptosis, late apoptosis, and necrosis, utilizing a two- color staining regime.

Quantitation of caspase activity is another popular means of determining what stage of apoptosis may be occurring. Caspases are protein-cleaving enzymes which are instrumental in the sequential disassembly of cells. BioSource International, Inc. has formulated kits for screening cell lysates for Caspase-2, -3, -6, -8, and -9 activity by either fluorometric or colorimetric detection. Also available are antibodies for Caspase-1, -3, -6, -8 and -10 for use in Western blotting.

A novel method for detection of apoptosis by flow cytometry is the PARP-FITC Cleavage Site-Specific Antibody (CSSA). This FITC-conjugated anti-PARP antibody specifically recognizes the 85kDa fragment of cleaved PARP which can be an excellent marker for detecting apoptosis. Along with the PARP-FITC CSSA, BioSource International, Inc. also provides an unconjugated and Biotin form of the antibody, and has formulated a PARP-FITC CSSA Kit which includes fixation and permeabilization buffers and matching peptide.

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