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. Author manuscript; available in PMC: 2014 May 14.
Published in final edited form as: J Immunol. 1999 Feb 1;162(3):1466–1479.

FIGURE 3.

FIGURE 3

Flow cytometric analysis of ROI production and mitochondrial transmembrane potential (ΔΨm) in PBL. A, PBL, freshly purified or prestimulated with Con A for 2 days (Con A, 2d), were incubated with 1 μg/ml Fas mAb CH-11 for 20 min on ice or 3 h at 37°C. As controls, freshly isolated PBL stimulated with Con A for 20 min on ice or 3 h at 37°C were also examined. Dead cells and debris were gated out by FSC/SSC measurements. ROI production was assessed by DHR fluorescence (mean channel, FL-1) of annexin V-PE (FL-2)-negative cells. Open curves correspond to control cells, while shaded curves represent Con A- and/or Fas-treated cells, as indicated for each histogram. The x-axis shows the log FL-1 fluorescence intensity; the y-axis indicates the cell number. Δψm was measured by DiOC6 fluorescence (FL-1) of annexin V-PE-negative cells or JC-1 fluorescence (FL-2) of live cells based on forward/side scatter (FSC/SSC) gating. Values over curves indicate the mean channels of DHR, DiOC6, and JC-1 fluorescence. B, Fas-induced mitochondrial ROI production in PBL prestimulated with 5 μg/ml Con A for 5 days. After prestimulation with Con A, PBL were incubated with 1 μg/ml Fas Ab CH-11. ROI production was assessed by DHR fluorescence (FL-1). PS externalization was determined by annexin V-PE staining (FL-2). Column 1 shows the percentage of annexin V-PE-negative cells and their right shift on the FL-1 axis. Column 2 shows histogram and mean channel number of DHR fluorescence of annexin V-PE-negative cells. T cells and their CD4 and CD8 subsets were identified by staining with Quantum Red-conjugated CD3, CD4, and CD8 Abs, respectively (FL-3). Columns 3–5 indicate the percentages of CD3-, CD4-, and CD8-stained cells and their mean DHR fluorescence (in parentheses) gated on annexin V-PE-negative cells. C, Fas-induced ΔΨm changes in PBL prestimulated with Con A for 5 days. Column 1 shows increased DiOC6 (FL-1) fluorescence of both CD4− and CD4+ compartments of PBL (FL-3) gated on annexin V-PE-negative cells in response to Fas stimulation. Column 2 shows the percentage of annexin V-PE-positive cells and increased DiOC6 fluorescence (right shift) of annexin V-negative cells. Column 3 indicates the histogram and mean channel of DiOC6 fluorescence gated on annexin V-PE-negative cells. Column 4 shows the histogram and mean channel of JC-1 fluorescence (FL-2) of live cells based on FSC/SSC gating. Data are representative of five independent experiments.