Assays for Apoptosis

Understanding the kinetics of cell death in your model system is critical. Some proteins, such as caspases, are expressed only transiently. Cultured cells undergoing apoptosis in vitro will eventually undergo secondary necrosis. 

Apoptotic cells in any system can die and disappear relatively quickly. The time from initiation of apoptosis to completion can occur as quickly as 2-3 hr. Therefore a false negative can occur if the assay is done too early or too late. Moreover, apoptosis can occur at low frequency or in specific sites within organs, tissues, and cultures. In such cases, the ability to rapidly survey large areas could be useful. In general, if detailed information on the mechanism of cell death is desired, the duration of toxin exposure, the concentration of the test compound, and the choice of assay endpoint become critical. 

A detailed description of all methodologies and assays for detecting apoptosis is beyond the scope of this chapter. However, some of the most commonly employed assays are mentioned and briefly described. Apoptosis assays, based on methodology, can be classified into six major groups, and a subset of the available assays in each group is indicated and briefly discussed  

Detection of caspases, cleaved substrates, regulators and inhibitors 

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V8. Vermes, I., Haanen, C., Steffens-Nakken, H., and Reutelingsperger, C. R, A novel assay for apoptosis Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. J. Immunol. Methods 184, 39-52 (1995). 

Annexin V Annexin V is a molecule that binds to phosphatidylserine and, therefore, if conjugated to a fluorochrome, will identify apoptotic cells (which express phosphatidylserine on their surface). In the assay for apoptosis, annexin V must be used in conjunction with propidium iodide in order to exclude dead cells (which express phosphatidylserine on the internal side of their cytoplasmic membranes). 

Analogous to cell viability measurements, many of the same basic concepts apply to the development of cell-based assays for apoptosis. The length of incubation of cells with the test compound is among the most important issues to address and optimize. The length of incubation is important because the markers of apoptosis may be present for relatively brief transient periods and subsequently disappear as the population of cells undergoes secondary necrosis. The induction of measurable caspase activity can occur in only a few minutes or can take days, depending on the model cell line, type of inducer, and effective concentration inside the cells. 

Ferhni, C. Scambia, G. Assay for apoptosis using the mitochondrial probes, Rhodaminel23 and 10-N-nonyl acridine orange. Nat. Protoc. 2007, 2, 3111-3114. 

Wronski, R. Golob, N. Grygar, E. Windisch, M. Two-color, fluorescence-based microplate assay for apoptosis detection. BioTechniques 2002, 32, 666-668. 

C. Bernstein, H. Bernstein, H. Garewal, P. Dinning, R. Jabi, R. E. Sampliner, M. K. McCuskey, M. Panda, D. J. Roe, L. L Heureux and C. Payne, A bile-acid-induced apoptosis assay for colon cancer risk and associated quality control studies. Cancer Res., 1999, 59(10), 2353. 

D5. Donner, K. J., Becker, K. M., Hissong, B. D., and Ahmed, S. A., Comparison of multiple assays for kinetic detection of apoptosis in thymocytes exposed to dexamethasone or diethylstilbesterol. 

Fig. I. Two-dimensional light scatter assay. Burkitt lymphoma cells induced into apoptosis by incubation with a MAb against IgM were assayed for forward light scatter (FSC) and 90° light scatter (SSC). Region 1, viable cells Region 2, apoptotic cells.

Fig. 5. Comparison of two-dimensional light scatter assay with subdiploid DNA peak assay. Burkitt lymphoma cells were serum-deprived for 14 d and then assayed for (A) forward light scatter (FSC) versus 90° light scatter (SSC) or (B) cell cycle. Note that although all the cells are clearly apoptotic as shown by the light scatter assay, there is only a small subdiploid peak (SD) present in the cell-cycle analysis, demonstrating that in this case, celt-cycle analysis alone would grossly underestimate the extent of apoptosis present in the cell population.

Bardales RH, Hailey LS, Xie SS, Schaefer RF, Hsu SM. In situ apoptosis assay for the detection of early acute myocardial infarction. Am J Pathol 1996 149 821-829. 

One of the main challenges in designing cytotoxicity and apoptosis assays for screening is determining an appropriate length of compound exposure. Difficulties arise because different classes of... 

Lobner D. 2000. Comparison of the LDH and MTT assays for quantifying cell death Validity for neuronal apoptosis J Neurosci Methods 96 147-152. 

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