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Discover hints and tips for successful apoptosis detection
There are a number of factors that need to be considered when trying to detect apoptosis. Here we review:
Timeline of events
Apoptosis typically progresses through the extrinsic or intrinsic death pathways, leading to activation of effector caspases, exposure of phosphatidylserine (PS) on the outer surface of the plasma membrane, cytochrome C release and genomic DNA fragmentation. The specific events that occur will depend on the apoptotic pathways that are activated. Moreover, the time required to detect different events or activities after apoptosis induction is often variable.
In a typical population, cells will be in different phases of the cell cycle, which means that individual cells within the same population will probably undergo apoptotic events at different times. For example, cells that have finished division will show G2/M arrest later than cells that have finished DNA replication (S phase). Hence, it is important to analyze single cells whenever possible to track activation of an apoptotic event.
Monitoring apoptotic events before or after the optimal time period will result in little or no signal, leading to the erroneous conclusion that the treatment did not induce apoptosis. Time course experiments are therefore essential to determine the optimal time to detect a given event.
Cell line variation
When choosing an apoptotic parameter to study, ensure that it is relevant to your experimental conditions. For example, the breast cancer cell line MCF-7 lacks a functional caspase 3 and therefore, determination of caspase activity using caspase 3 substrates or caspase 3 inhibitors will show no results.
Different cell lines may respond differently to identical conditions, and certain treatments may induce apoptosis in one cell line but not others. Perform an initial dose response curve to identify the appropriate concentration of a compound and timing of the treatment for best results in your cells of interest.
Published literature and protocols offer invaluable guidance but do not rely on them exclusively to set your experimental conditions. Subtle lab-to-lab differences may affect your results.
Apoptosis inducers and inhibitors
The use of inducers or inhibitors for specific steps of the apoptosis cascade, is essential in order to provide positive and negative controls. These ensure that the assay worked and confirm that apoptosis is actually happening.
The table below highlights known apoptotic inducers and inhibitors for functions linked to cell death:
|Apoptosis Activator 2 (AAII)||Cytochrome C, caspase activator and apoptosis inducer|
|Cycloheximide||Protein synthesis inhibitor|
|Mitomycin C (MMC)||Anticancer and antibiotic agent|
|Oxaliplatin / cisplatin||Platinum anticancer agent. Induced DNA cross-linking.|
|CPT 11 (Irinotecan)||Potent DNA topoisomerase inhibitor|
|Camptothecin||DNA topoisomerase inhibitor|
|FCCP||Mitochondrial oxidative phosphorylation inhibitor|
|z-VAD-(OMe)-FMK||Cell permeable, irreversible general caspase inhibitor|
|z-D(OMe)E(OMe)VD(Ome)-FMK||Cell permeable caspase 3 inhibitor|
|z-IETD-FMK||Caspase 8 inhibitor|
|Pifithrin-μ||Inhibitor of p53-mediated apoptosis|