Caspase-3-Colorimetric-Assay-Kit-[CPP32]-From-Chemicon

Apoptosis is a highly regulated process with important roles in organogenesis, turning off immune responses and elimination of damaged or modified cells that pose a risk to tissue homeostasis. A proper assay of this process is essential considering that imbalanced apoptosis levels result in significant pathology: A lack of response to apoptosis stimuli leads to cancer while excessive apoptosis induces degenerative processes. Generally, apoptosis can occur through either the intrinsic or the extrinsic pathway. The intrinsic pathway is induced by DNA damage, anoikis, or other stress (UV, heat shock, growth factor deprivation, etc). The main characteristic of the intrinsic pathway is the loss of mitochondrial membrane integrity, subsequent release of cytochrome c, APAF and Smac/DIABLO, followed by activation of caspase 9. The extrinsic pathway is triggered by the interaction of the ligands belonging to the TNF family with their cognate receptors. Cells undergoing apoptosis through this pathway are further subdivided into type I (characterized by important cleavage and activation of caspase 8), or type II. In type 2 cells, caspase 8 is cleaved in small amounts while proapoptotic BH3-only proteins are activated with subsequent permeation of the mitochondrial membrane and caspase 9 activation. Both pathways converge on activating effector caspases 3, 6 and 7, followed by cleavage of vital cellular structures and cellular demise.

Because caspase 3 is an effector caspase, determining caspase 3 activity reflects the level of apoptosis in the tested cells, irrespective of the inducing stimulus. To investigate the pathway involved in caspase 3 cleavage, one should determine the activation of initiator caspase 8, or caspase 9. Caspase 3 activation can be assessed either by detection of the cleaved caspase 3 with specific antibodies (Western blot), or by enzymatic assays already optimized in kits, such as the Caspase 3 Colorimetric Assay Kit [CPP32] from Chemicon. Quantification in enzymatic assays involves measuring the fluorescence, luminescence, or absorbance of a specific substrate cleaved by the caspase enzyme in the analyzed samples.

Chemicon’s Colorimetric Caspase 3 Assay Kit is based on caspase 3’s ability to recognize and cleave a substrate containing the DEVD motif. The protocol is straight forward: The activated caspase 3 in the lysed cells cleaves the para-nitro aniline (pNA)-DEVD substrate. pNA is subsequently determined by measuring absorbance at 405 nm and compared to a standard curve obtained by serial dilutions of the pNA standard provided in the kit. For good and consistent results, there are several important observations one should take into consideration when using this kit: Use fresh lysates, work quickly and on ice, plate sufficient numbers of cells/use sufficiently concentrated samples. Cells should be stimulated or incubated long enough after stimulation to allow significant caspase3 activation.

I found this kit very useful in my experiments as it saved me time when compared with Western blot-based assays for caspase 3 cleavage. Another advantage of this kit is that it does not require fluorescence or luminescence readers or fluorescence based imaging. It only employs a spectrophotometer or an ELISA plate reader with a filter for the above mentioned wavelength, which makes this kit suitable for most of the labs. One thing to keep in mind when using this type of kit is that samples should be run in duplicate; this should be considered when planning and purchasing the reagents required for the experiments.

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Stefan David, MD
Postdoctoral Fellow
Division of Gastroenterology/Department of Medicine
Johns Hopkins School of Medicine
United States