Apoptosis is a common phenomenon in eukaryotic cells and is integral to many fields of research, from developmental biology to infectious disease. There are many different morphological and biochemical changes that occur when cells undergo apoptosis and therefore, there are many methods of detecting apoptotic cells. In our lab, we combined the visualization of nuclear fragmentation using DAPI (a morphological change) with Caspase 9 cleavage (a biochemical change) using Calbiochem’s Caspase 9 Colorimetric Assay Kit. Our cells were human cell lines (HeLa and Caco-2) and we used various apoptotic stimuli, including staurosporine and different xenobiotics, such various bacterial factors. Previously, we had tried other ways of detecting apoptosis, such as Annexin V-FITC detection and Western blot analysis, but we found the Caspase 9 kit by Calbiochem to be the most sensitive and most reproducible. Also, Caspase 9, sometimes referred to as the apoptosome, is essential for the execution of apoptosis and acts very early on, and is thus a logical target for apoptotic detection.
The Calbiochem Caspase 9 kit is based on the amino acid sequence LEHD which is recognized by Caspase 9. LEHD is linked to the chromophore pnitroanilide (pNA) which when cleaved by Caspase 9 can be detected by spectrophotometry at about 400 nm. We have routinely used the kit with adherent cells although the kit protocol refers to cells in suspension. We modified the kit’s protocol for adherent cells and have had consistent success with confluent cells in a 24-well plate format. Our cells are treated experimentally and then lysed while still adherent, with the kit’s cell lysis buffer. The protein concentration of the lysate is quickly determined using Bradford reagent and then the lysate is mixed with reaction buffer containing the LEHD-pNA. This is then incubated at 37ºC for about 30 min in the dark (1 h appears to be too long, although it does say 1-2 h in the kit). Our samples are then measured at 400 nm in a 96-well plate format; a cuvette based format also works well. The kit needs to be stored at -20ºC, but we aliquot the reagents to avoid repeated freeze-thawing; we store opened aliquots in the dark at 4ºC for one to two weeks without any noticeable changes in results.
This kit was very sensitive. Our positive control (using Staurosporine) displayed huge Caspase-9 activation while our negative, untreated control displayed minimal Caspase 9 background. Also, the Caspase-9 signal supported results obtained with other apoptosis detection methods, such as nuclear fragmentation. The kit was easy to use and the instructions, easy to follow. All reagents were supplied in the kit and it was quick to perform, although measuring the lysate by Bradford and waiting for the reaction to occur at 37ºC will add about 1 h in total onto the procedure. The reagents were not compromised by exposure to bacteria or bacterial products and it was also noted that the age or confluency of the cell lines had little effect on Caspase-9 activation. I would recommend this kit as it has given us the most success in measuring apoptosis and is highly sensitive, giving a quantitative output of Caspase-9 activity with good reproducibility.