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e-Gsi Sdiviiy {FOG)

Figure 7 Correlation of gene expression with cell cycle progression. (A) The /3-galactosidase gene expression in a Jurkat T cell line is controlled by DNA enhancer elements bound by a nuclear transcription factor termed NF-AT. Stimulation of T cells results in a signal transduction cascade, including the activation of this nuclear factor. (B) After stimulation for various periods of time up to 12 h, the fraction of cells expressing /3-galactosidase activity (and thus having activated NF-AT) increases. 0-Galactosidase activity is measured using the fluorogenic substrate fluorescein di-galactoside (FDG). The activation signal is a threshold event: after activation, no further increase in the production of /3-galactosidase occurs (compare the positive cells at 2 h with those at 12 h). (C) Simultaneous quantitation of the activation of NF-AT (as revealed by /3-galactosidase activity) and cell cycle progression (as revealed by the DNA content of cells) after 2 h of stimulation. Note that cells that have activated NF-AT are more likely to already be in cell cycle progression (S + G2/M) than unactivated cells. (Courtesy of Dr Stephen Fiering.)

to gene expression analysis the power of multiparameter FACS; that is, the quantitation of different gene elements within individual cells, providing a correlated distribution of expression of those genes in a population (for example, see Figure 8).

The FACS is also a powerful tool for measuring the physiological state of cells. A commonly measured physiological response is the cytoplasmic calcium concentration. Calcium is mobilized from internal stores as well as the extracellular milieu in response to mitogenic signals. Thus, measuring the change in the internal calcium concentration after signaling is an important indicator of the responsiveness of cells, and a predictor of their ultimate proliferative capacity. Figure 9 shows an example of such an experiment. This experiment demonstrates that peripheral blood T cells have a heterogeneous responsiveness in terms of the ability to flux calcium - a heterogeneity which is only evident by combining this experiment with antibody stains to subdivide the populations.

It is very common in flow cytometric analysis to quantitate live versus dead cells. In general, this is most easily accomplished using propidium iodide (PI). PI binds avidly to DNA, but is excluded from DNA in live cells because it cannot cross the mem-

Bex GFP

Figure 8 Simultaneous detection of two mutants of green fluorescent protein. Two different mutants (Bex and Vex) of the inherently-fluorescent GFP were selected so that they could be simultaneously and independently measured. In this experiment, cells were infected with a mixture of two different viruses, each expressing one of the mutant GFPs. Most cells were uninfected (expressing neither GFP); some cells were singly infected; and some cells were infected by both viruses (and show expression of both the Bex and Vex mutant GFPs). (Courtesy of Dr Michael Anderson.)

Bex GFP

Figure 8 Simultaneous detection of two mutants of green fluorescent protein. Two different mutants (Bex and Vex) of the inherently-fluorescent GFP were selected so that they could be simultaneously and independently measured. In this experiment, cells were infected with a mixture of two different viruses, each expressing one of the mutant GFPs. Most cells were uninfected (expressing neither GFP); some cells were singly infected; and some cells were infected by both viruses (and show expression of both the Bex and Vex mutant GFPs). (Courtesy of Dr Michael Anderson.)

Time after stimulation (min)

Figure 9 T cell signal transduction measured by calcium translocation. T cells isolated from a healthy individual were loaded' with lndo-1, a fluorescent molecule whose spectrum depends on calcium concentration. By measuring and calculating the ratio of two different lndo-1 fluorescences, one which declines and one which increases with increased calcium concentration, an estimate of intracellular calcium content can be made. In this experiment, T cells were also surface stained to determine their differentiation state. They were then stimulated through the T cell receptor, the primary signal for activation of these cells. The figure shows the relative calcium content after stimulating CD4 T cells with antibody to CD3 (dark lines). Cells that coexpress CD45RA and CD62L (and are 'naive' T cells) have a greater capacity to translocate calcium into the cytoplasm, compared with the memory subsets. This observation correlates with the greater capacity of naive T cells to proliferate after such a stimulation. The heterogeneous calcium flux is due to a difference in the CD3 signal transduction pathway: when the cells are stimulated with low concentrations of ionomycin (light lines), the three subsets have indistinguishable calcium mobilization profiles.

brane. Dead cells, on the other hand, have permeable membranes that readily allow PI to enter and bind to the DNA. Thus, inclusion of a small amount of PI in all samples allows dead cells to be excluded from further analysis (or at least separately enumerated). Recently, much interest has focused on pathways by which cells die, in particular that of programmed cell death (or apoptosis). Several assays exist for the quantitation of apoptosis; such an assay is shown in Figure 10. In this example, the cells that have been signaled to die are shown to progress through several distinct stages.

In addition to these examples, assays that measure intracellular pH, reduction-oxidation (redox) potential, mitochondrial activity, RNA content, membrane area, membrane potential and endocytic activity have been developed for use with the multiparamet-ric paradigm of flow cytometry. Changes in these measurements reveal signal transduction events, stress responses, mitogenic responses, etc. -important parameters of the functional capacity of cells. While most of these are used primarily in basic

Annexin V binding

Figure 10 Discrimination of dying and apoptotic cells. AnnexinV is a protein which binds to phosphatidylserine (PTS). PTS is normally not expressed on the outside of cellular membranes, but only on the inside. When cells commit to apoptosis (programmed cell death), one of the earliest events is the loss of this membrane asymmetry in PTS expression. Using fluorescently-conjugated Annexin V, cells early in the apoptosis commitment phase can be identified. During later stages of cell death, the chromatin condenses and binds Hoechst 33342 to a very high degree (note this use in distinction to Figure 7, where quantitative binding reveals DNA content); in addition, the Hoechst dye itself is no longer actively removed from the cells. In this figure, Jurkat T cells were induced to apoptosis; after various times they were assayed for Annexin V binding and Hoechst 33342 uptake. There is a clear progression of cells from double-negative, to Annexin V-positive (apoptosis-committed), to double-positive (dead). (Courtesy of Dr Peter Katsikis.)

Annexin V binding

Figure 10 Discrimination of dying and apoptotic cells. AnnexinV is a protein which binds to phosphatidylserine (PTS). PTS is normally not expressed on the outside of cellular membranes, but only on the inside. When cells commit to apoptosis (programmed cell death), one of the earliest events is the loss of this membrane asymmetry in PTS expression. Using fluorescently-conjugated Annexin V, cells early in the apoptosis commitment phase can be identified. During later stages of cell death, the chromatin condenses and binds Hoechst 33342 to a very high degree (note this use in distinction to Figure 7, where quantitative binding reveals DNA content); in addition, the Hoechst dye itself is no longer actively removed from the cells. In this figure, Jurkat T cells were induced to apoptosis; after various times they were assayed for Annexin V binding and Hoechst 33342 uptake. There is a clear progression of cells from double-negative, to Annexin V-positive (apoptosis-committed), to double-positive (dead). (Courtesy of Dr Peter Katsikis.)

research, some assays are beginning to find clinical relevance.

For example, oxidative stress has been shown to accompany HIV disease. A FACS assay which can measure intracellular glutathione (the major intracellular antioxidant) showed that lymphocytes in persons with HIV disease are depleted for glutathione - and that this depletion was a predictor of survival in advanced stage disease (Figure 11, see p. 942). The combination of surface immunophenotyp-ing and a physiological measurement therefore provides a powerful, clinically relevant parameter. Figure 11 also shows an example of the in vivo quantitation of gene expression of FACS. In this case, the number of CD38 molecules per CD8 T cell (i.e. proportional to the fluorescence of cells stained with phycoerythrin-conjugated anti-CD38 monoclonal antibody) also provides a significant predictor of progression to death in AIDS. Using these two parameters simultaneously is the most significant predictor of lifespan in AIDS patients known to date - far more significant that CD4 T cell counts, the most commonly used surrogate marker.

Clinical relevance has also been ascribed to the quantitative determination of DNA content in lymphocytes. In this case, lymphocyte membranes are disrupted to allow free entry of dyes such as Hoechst 33342 or PI. Quantitative fluorescence measurements easily reveal 2N or 4N DNA content (akin to the example in Figure 7). Clinical relevance comes from the observation that many leukemias and lymphomas have aberrant DNA content (aneuploidy). There is an extensive literature on typing such cancers based on DNA quantification.

Finally, the FACS can be used to perform powerful functional assays. FACS-based assays exist which can measure activation, proliferation, cytotoxic activity, apoptosis and cytokine production. Often, the FACS assays are considerably more sensitive than bulk assays. For instance, cytokine production assays can be performed in cell populations stimulated for only 6 h (for example, see Figure 12, see p. 942). Bulk assays, which measure cytokine production in supernatant, are technically easier to perform but yield considerably less information, together with greater potential artifact. Bulk assays for cytokine

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Days since mitral measurement

Figure 11 FACS-measured quantities are powerful prognostic variables In AIDS. Shown are Kaplan-Meier survival plots, which predict the survival of a group of individuals based on a measurement made at an initial time (day 0). (Left) The amount of glutathione (GSB) (the major intracellular antioxidant) was measured in CD4 T cells in 59 HIV-infected adults with advanced disease. Those with high glutathione were far more likely to survive 2 years than those with low glutathione. (Middle) The same individuals were divided on the basis of CD38 expression on CD8 T cells. Quantltating the absolute expression of this molecule, which may reflect the activation state of the immune system, also provides a powerful prognostic of death. (Right) The glutathione measurement and CD38 measurement were correlated to provide an index of individuals who are at high risk for progression to death (i.e. low glutathione and high CD38) versus those who are at low risk. The combination of these two parameters provides the most powerful prognostic value for predicting survival in AIDS patients.

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