FLOW CYTOMETRY-BASED ASSAYS

Flow cytometers, which are widely used in the study of lymphocytes, offer several advantages in the study of apoptosis. These include the ability to analyze multiple parameters simultaneously and the possibility of analyzing up to 20,000 or more individual cells in each sample.

Altered Cell Size

One of the distinguishing features of apoptosis is cell shrinkage.¹¹⁴ This change has been attributed to potassium ion loss¹¹⁵ that occurs, at least in part, as a consequence of caspase-dependent activation of the Kv1.3 potassium channel¹¹⁶ and degradation of the plasma membrane Na/K-ATPase.¹¹⁵ Because larger cells scatter light more than smaller cells, the decrease in cell size can be monitored by changes in light scatter properties. These observations provide the basis for studies that use changes in light scatter to monitor apoptosis.

To apply this approach, cells treated with a proapoptotic stimulus for varying lengths of time are subjected to flow cytometry and are monitored for forward scatter, as illustrated in Figure 3.1E. This assay can be performed on either unfixed or fixed cells. In either case, apoptotic cells display less forward scatter than control cells.

Although this assay is rapid and simple, it has several limitations. Because each cell can give rise to multiple apoptotic bodies, merely quantifying the number of small particles can potentially result in overestimation of the number of cells that have undergone apoptosis, particularly at later time points when cells have fragmented.

Loss of Mitochondrial Transmembrane Potential (ΔΨ_m)

During the last decade, there has been considerable interest in mitochondrial changes during apoptosis.⁷³ This interest was initially spurred by claims that loss of mitochondrial transmembrane potential was a particularly early event in apoptosis¹¹⁸,¹¹⁹ and by demonstration that cytochrome c played a critical role in activation of the Apaf-1/caspase-9 pathway.⁷³,¹²⁰ At one time, it was thought that loss of mitochondrial transmembrane potential might reflect inhibition of electron transport and concomitant diminution of proton translocation as a consequence of cytochrome c release. Subsequent experiments have shown that loss of ∆Ψ_m occurs after cytochrome c release and likely reflects caspase-mediated cleavage of another component of the electron transport chain.^121,122 Despite the fact that loss of ∆Ψ_m represents a late event in apoptosis, there remains considerable interest in its measurement.

The electrochemical potential difference across the inner mitochondrial membrane ordinarily drives mitochondrial ATP synthesis but can also be used to promote accumulation of membrane­permeant cationic dyes against a concentration gradient. The extent to which these dyes are concentrated in isolated mitochondria is directly related to the magnitude of the electrochemical gradient.¹²³ These observations form the basis for flow cytometry-based methods of assessing ∆Ψ_m.

To apply this approach, cells treated with an apoptotic stimulus are incubated briefly with membrane-permeant cationic dyes (e.g., 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolcarbocy- anine iodide or 3,3-dihexyloxacarbocynine iodide) and are immediately subjected to flow cytometry. Cells containing a normal ∆Ψ_m accumulate larger amounts of these dyes than cells with diminished mitochondrial transmembrane potential.

The present approach can measure transmembrane potential relatively simply and easily in 20,000 or more cells/sample. It is important, however, to emphasize several limitations of this method. First, although it has been suggested that altered ∆Ψ_m might be a highly reliable early marker of apoptosis,¹¹⁸,¹¹⁹,¹²⁴ this change is not universally observed.¹²⁵ Second, this change is not specific for apoptosis. Instead, decreased ∆Ψ_m is also extensively documented in cells undergoing necrosis¹²⁶ and can result from any treatment that alters mitochondrial electron transport or proton translocation (e.g., metabolic poisons such as 2,4-dinitrophenol or sodium azide). Third, the quan­tum yield of many of the cationic dyes used to measure mitochondrial transmembrane potential is diminished by p-aminobenzoic acid derivatives that quench the fluorescent emission.¹²⁷ Thus, changes in fluorescent emission do not always reflect altered ∆Ψ_m and certainly do not reflect alterations specific to apoptosis.

Dye Uptake

A number of techniques for detecting apoptosis rely on changes in dye uptake that can be measured by flow cytometry. For example, cells can be incubated in Hoechst 33342 or 7-aminoactinomycin D (7-AAD) at 4°C and then subjected to flow microfluorimetry. Ordinarily, Hoechst 33342 and 7-AAD are excluded from cells at 4°C. Cells that have ruptured readily take up these agents and, therefore, fluoresce strongly.

Because Hoechst 33342 and 7-AAD freely penetrate the plasma membrane above its phase transition temperature, which is typically at 20°C, it is critical that cells be kept as close to 4°C as possible during exposure to the dye. When this precaution is achieved, the technique provides a straightforward means of assessing dye permeability in thousands of individual cells per sample. In addition, if the dyes listed above are combined with fluorochrome-labeled antibodies with suitable spectral properties, apoptosis can be readily quantified in a subset of cells (e.g., CD4-expressing lymphocytes) in a larger cell population.

Despite these advantages, two potential limitations of this approach should be kept in mind. First, when this approach is used to evaluate apoptosis in rare cell subsets, it can be difficult to use other techniques (morphology, agarose gel electrophoresis) to confirm that the cells taking up dye are truly undergoing apoptosis. In addition, it is not entirely clear that intermediate dye uptake is absolutely specific for apoptosis. It is conceivable that other changes within cells (e.g., ATP depletion or altered plasma membrane lipid composition) might also allow penetration of small amounts of these dyes into cells. As long as these potential limitations are kept in mind, dye uptake assays seem to offer a rapid and powerful technique for investigating apoptosis in mixed populations of cells.

ACKNOWLEDGMENTS

Research in the authors’ laboratory is supported by NIH grant CA69008. We thank Son Le, William C. Earnshaw, Greg Gores, David Vaux, and Yuri Lazebnik for spirited discussions; David Loegering, Karen Flatten, and Julie Wahlstrand for technical assistance with the experiments illustrated in Figure 3.1; and Deb Strauss for editorial assistance.

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