Aging Aging

78 Carmody et al.Fig. 3. Annexin V/PI dual staining of human T cells treated with ActinomycinD. Jurkat cells were treated for 4 h with 15 mg/mL of Actinomycin D. Cells werestained with Annexin V-FITC (FL-1, y-axis) and PI (FL-2, x-axis) as described inSubheading 2.1. Normal cells are negative for both Annexin and PI and appear inthe lower left quadrant. Apoptotic cells with intact membranes stain with Annexin,but not with PI and therefore appear in the upper left quadrant. Primary and secondarynecrotic cells have disrupted membranes and stain with both Annexin Vand PI (upper right quadrant).4. Wash twice in PBS and keep on ice and in the dark until read on a flowcytometer.5. Measure green fluorescence (labeled DNA strand breaks) following excitationat 488 nm using a 525 nm band pass filter (FL-1, log scale; see Subheading 3.and Table 1).3.2.1. Analysis of Cell Cycle in Conjunction with TUNEL AssayAfter the TUNEL assay procedure has been completed resuspend cells in1mLof PBS containing 5 µg/mL of PI, and 0.1% DNase-free RNase A. Cellcycle analysis can then be carried by measuring the red fluorescence of PI at>600 nm (FL-2, linear scale; see Subheading 3. and Table 1) (Fig. 4-1).3.2.2. Analysis of Antigen Expression in Conjunctionwith TUNEL AssayThe combination of TUNEL staining with immunofluorescence labeling ofa cell-specific antigen allows the subsequent analysis of apoptosis in specificsubpopulations. Cells may be labeled for antigen expression prior to or followingfixation/permeabilization depending on the nature of the epitope. It is recommendedthat it first be determined whether epitope labeling is affected bythe fixation/permeabilization process. The following procedure detects an intra-