Figure 6.
Figure 6. U-266-1970 cells expressing high levels of Fas are more sensitive to Fas-mediated apoptosis. (A) FACS analysis of Fas expression in living cells. U-266-1970 cells were preincubated with IL-6 (20 U/mL) alone (solid line) or in combination with IFN-γ (1000 U/mL; dotted line) for 96 hours as indicated, followed by a 24-hour incubation with agonistic anti-Fas antibody CH-11 or isotype-specific control IgM. The level of Fas expression in annexin V-/PI- cells was analyzed in 4 independent experiments using flow cytometry. One representative analysis is shown. (B) Analysis of Fas-mediated apoptosis in cells sorted according to Fas expression. U-266-1970 cells expressing high or low levels of Fas were separated using FACS and allowed to recover for 4 days. The sorted cells were incubated for 24 hours with agonistic anti-Fas antibody CH-11 or isotype-specific control IgM. The graph shows Fas-expression and annexin V expression in PI- cells, evaluated by flow cytometry as described. The horizontal line indicates the MFI of the total population of U-266-1970 cells prior to CH-11 stimulation. Four experiments were performed. The percentage of Fas+ cells from one representative experiment is shown. (C) Expression of Fas in transfected cells. U-266-1970 cells were cotransfected with EGFP and either empty pcDNA3 vector or a vector encoding CD95 (pcDNA-FAS), allowed to recover for 24 hours, and then subjected to FACS analysis of Fas expression as described in “Patients, materials, and methods.” The diagram shows the relative expression of Fas in EGFP+, transfected cells as fold induction of MFI. (D) Fas-mediated apoptosis in transfected cells. At 24 hours after transfection, cells were stimulated with agonistic anti-Fas antibody CH-11 or isotype-specific control IgM for 24 hours. The proportion of annexin V+/PI- cells was determined by flow cytometry as described in “Patients, materials, and methods.” The graph shows the percentage of apoptotic cells. Mean values ± SD (n = 3). The asterisk indicates significant differences to the corresponding control values (P < .05).

U-266-1970 cells expressing high levels of Fas are more sensitive to Fas-mediated apoptosis. (A) FACS analysis of Fas expression in living cells. U-266-1970 cells were preincubated with IL-6 (20 U/mL) alone (solid line) or in combination with IFN-γ (1000 U/mL; dotted line) for 96 hours as indicated, followed by a 24-hour incubation with agonistic anti-Fas antibody CH-11 or isotype-specific control IgM. The level of Fas expression in annexin V-/PI- cells was analyzed in 4 independent experiments using flow cytometry. One representative analysis is shown. (B) Analysis of Fas-mediated apoptosis in cells sorted according to Fas expression. U-266-1970 cells expressing high or low levels of Fas were separated using FACS and allowed to recover for 4 days. The sorted cells were incubated for 24 hours with agonistic anti-Fas antibody CH-11 or isotype-specific control IgM. The graph shows Fas-expression and annexin V expression in PI- cells, evaluated by flow cytometry as described. The horizontal line indicates the MFI of the total population of U-266-1970 cells prior to CH-11 stimulation. Four experiments were performed. The percentage of Fas+ cells from one representative experiment is shown. (C) Expression of Fas in transfected cells. U-266-1970 cells were cotransfected with EGFP and either empty pcDNA3 vector or a vector encoding CD95 (pcDNA-FAS), allowed to recover for 24 hours, and then subjected to FACS analysis of Fas expression as described in “Patients, materials, and methods.” The diagram shows the relative expression of Fas in EGFP+, transfected cells as fold induction of MFI. (D) Fas-mediated apoptosis in transfected cells. At 24 hours after transfection, cells were stimulated with agonistic anti-Fas antibody CH-11 or isotype-specific control IgM for 24 hours. The proportion of annexin V+/PI- cells was determined by flow cytometry as described in “Patients, materials, and methods.” The graph shows the percentage of apoptotic cells. Mean values ± SD (n = 3). The asterisk indicates significant differences to the corresponding control values (P < .05).

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