Flow cytometry cell death

Alternatively, or in parallel, propidium iodide staining of cells and flow cytometry detection measures the DNA content of the cells and distinguishes between cells in different cell cycle phases (e.g., S-phase and Gi phase). It also measures cell death, and to some extent the appearance of abnormal cells can be detected. 

Buenz, E. J. Limburg, P. J. Howe, C. L. A high-throughput 3-parameter flow cytometry-based cell death assay. Cytometry 2007, 71A, 170-173. 

Rituximab is a monoclonal antibody to the CD20 receptor expressed on the surface of B lymphocytes the presence of the antibody is determined during flow cytometry of the tumor cells. Cell death results from antibody-dependent cellular cytotoxicity. The pharmacokinetics of rituximab are best described by a two-compartment model, with a terminal half-life of 76 hours after the first infusion and a terminal half-life of 205 hours after the fourth dose.36 Rituximab has shown clinical activity in the treatment of B-cell lymphomas that are CD20+. Side effects include hypersensitivity reactions, hypotension, fevers, chills, rash, headache, and mild nausea and vomiting. 

Vermes, I., Haanen, C., and Reutelingsperger, C., Flow cytometry of apoptotic cell death, J. Immunol. Methods, 243, 167, 2000. 

In this study, annexin V/PI assay was used to detect cell death by flow cytometry (30) (see Note 11). 

Different methods can be used to detect cell death in CLL cells. Methods based on detection of cell death by flow cytometry are recommended as CLL cells are easily distinguishable by forward and size scatter from possible contamination of... 

The only difficulty in using flow cytometry to monitor cell death is that, as mentioned in Chapter 3, dead cells have different scatter properties than living cells. In particular, because of their perforated outer membrane, they have a lower refractive index than living cells and therefore have forward scatter signals of lower intensity. For this reason, it is important not to use a gate or forward scatter threshold when analyzing a population for the proportion of dead and live cells. Any forward versus side scatter gate drawn around normal lymphocytes, for example, will always show most if not all of the cells... 

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. 

Information gained from assays and future potential So far, few field measurements have been made of caspase-like activities (Berman-Frank et al., 2004 Vardi et al., 1999), but the assay methods appear to be sensitive enough to allow use in natural communities. As work using cultures proceeds, and our understanding of cell death processes improves, assays of capase-like activity may offer an important means to distinguish different forms of cell mortality. Aside from bulk in vitro assays, the availability of ceU-permeable substrates, coupled with flow cytometry will provide improved resolution and specificity (e.g. Bidle and Bender, 2008). 

Dive, C Gregory, C. D., Phipps, D. J., Evans, D. L., Milner, A. E and Wyllie, A. H. (1992) Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multi parameter flow cytometry. Biochim. Biophys. Acta. 1133,275-285. 

Many of the cell-death assays described above are readily performed by flow cytometry... 

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... 

Figure 4. A) Growth curves of PARG MEFs. Cells from littermates were seeded at a density of 10 cells/well in 6-well plates and counted every 2 days. B) Analysis of cell death and cell cycle progression after y-irradiations. Cells were irradiated at 10 Gy and the sub-G 1 population was analyz by flow cytometry (FL2H channel).

Apoptosis is a dynamic physiological process in the normal development and homeostasis of multicellular organisms. The ability to monitor cell death events over time provides a unique tool for the evaluation of the therapeutic efficacy of experimental therapeutic agents and for studies on the role of apoptosis in various disease processes. Conventionally, apoptosis in a population is determined at a specific time by microscopic examination or flow cytometry. However, apo-ptotic cells exist in vitro for a hmited time and occur only in a relatively narrow temporal window, after which they promptly disintegrate. Thus, an accurate determination of apoptosis in a population of cells requires either frequent determinations or continuous monitoring (Fig. 4). 

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