Flow cytometry apoptosis assay

Christensen, M.E., Jansen, E.S., Sanchez, W., Waterhouse, N.J., 2013. Flow cytometry based assays for the measurement of apoptosis-associated mitochondrial membrane depolarisation and cytochrome c release. Methods 61, 138-145. 

Four methods of quantitating apoptosis by flow cytometry are described in this chapter. The two-dimensional light scatter assay measures the reduction in cellular volume and increase in cell density that are observed during apoptosis and that are revealed by a decrease in forward light scatter and an increase in... 

Gorczyca, W, Melamed, M. R., and Darzyrikiewicz, Z. (1993) Apoptosis of S-phase HL-60 cells induced by topoisomerase inhibitors detection of DNA strand breaks by flow cytometry using the in situ nick translation assay Toxicol Lett 67, 249-258. 

Apoptosis Apoptosis is an ordered, active process that brings about the death of a cell as an important part of the maintenance of organismal homeostasis. Apoptosis can be assayed, in flow cytometry, by, for example, looking at the expression of phosphatidylserine on the cell surface, by looking for nuclei with less-than-normal (sub-GO/Gl) amounts of DNA, and by looking for an increase in DNA fragment termini. 

As with flow cytometry, multiparameter apoptosis assays may also be performed by confocal laser scanning microscopy (CLSM). Using the approach similar to that detailed above for flow cytometry, we have examined NADPH content, mitochondrial membrane potential (CMX Rosamine fluorescence), and mitochondrial mass (Mitotracker Green), by CLSM. Figure 3 shows an example of a typical multiparameter assay performed by confocal microscopy. 

As indicated earlier, there are many different parameters that can be used to measure apoptosis by flow cytometry. We have chosen two assays of... 

Cell-Based Bioassay For a cell-based Nab bioassay, treated cells respond directly or indirectly to the drug in a concentration-dependent manner. Possible biological responses of the cells to chug treatment include cell proliferation, apoptosis, phosphorylation, chemokine release, and expression of proteins or genes [15,22,28 30]. These responses may by quantitated by techniques such as immunoassay, multiplex assays [31], flow cytometry [23], and gene expression profiling [32]. 

A number of methods for the study of apoptosis and necrosis by flow cytometry have been developed (see review by Steensma et al., 2003). Unfortunately, none of the methods are rigorously specific for apoptosis and many show poor overall specificity for cell death or cannot discriminate between the terminal stages of apoptosis and necrosis. Moreover, some of the fluorochromes used to detect apoptosis have emission spectra that fully overlap the spectra of those typically used for immunophenotyping or have a broad emission spectrum that makes compensation in multi- or polychromatic flow cytometry difficult, if not impossible. Thus, the preferred method would be one that measures two aspects of apoptosis, can discriminate between apoptotic and dead cells, and uses fluorochromes that allow simultaneous immunophenotyping. One such method is the annexin-V/7-amino-actinomycin-D assay described by Merchant et al. (2001) that is available in kit from BD Biosystems (San... 

Flow cytometry has been used successfully for analysis of apoptosis. Annexin V/Propidium iodide assay in this technique can differentiate between dead and live cells.This assay has been shown to provide a single-cell-based, rapid, quantitative, and sensitive assay to detect lymphocyte-mediated target cell killing in various animal models. Unlike conventional chromium release assays, the FC assay enables monitoring of cellular immune responses in real time and at the single-cell level using diverse fluorescence detection methods such as flow cytometry and fluorescence and CLSM [48]. 

BAB 14] Babu U.S., Gaines D.M., Wu Y. et al., Use of flow cytometry in an apoptosis assay to determine pH and temperature stabihty of shiga-like toxin 1 ,... 

Chapman, R. S., Chresta, C. M Herberg, A. A., Beere, H. M., Heer, S., Whetton, A. D Hickman, J. A., and Dive, C. (1995) Further characterisation of the in situ terminal deoxynucleotidyl transferase (TdT) assay for the flow cytometric analysis of apoptosis in drag resistant and drug sensitive leukaemic cells. Cytometry 20,245-256. 

Hotz, M. A., Gong, J., Traganos, F., and Darzynkiewicz, Z. (1994) Flow cytometric detection of apoptosis comparison of the assays of in situ DNA degradation and chromatin changes. Cytometry 15(3), 237-244. 

---