Quantification of apoptotic and necrotic cells in patient and control fibroblasts. (A–D) Representative density plots showing the analysis of noninflammatory apoptotic and inflammatory necrotic cells by flow cytometry. (A and B) Cells were gated for single cells by FSC-H and FSC-A (A), followed by cell selection to exclude cell debris from the analysis with SSC-A and FSC-A (B). These events were then analyzed for the frequency of apoptotic cells (Annexin-V positive, SYTOX Green negative, C) and necrotic cells (Annexin-V negative, SYTOX Green positive). (D) Representative density plots showing the frequency of Annexin-V– and SYTOX Green–positive cells in primary fibroblasts from the patient (P, top) and two controls (C1, middle and C2, bottom) that were mock transfected (transf ctrl, left) or transfected with PolyIC (500 ng/ml, 24 h, right). (E–G) Graphs showing the frequency of inflammatory necrotic cells in primary fibroblasts from two controls (C, grey) and the patient (P, red). Cells were left untreated (UT) or treated with H₂O₂ (2 mM, 24 h, E), infected with mock virus (mock) or RSV (RSV-A strain long, MOI 3, 24 h, F), or mock transfected (transf ctrl) or transfected with PolyIC (500 ng/ml, 24 h, G). Bars show the mean ± SD values of two experiments (E–G) performed in duplicate and triplicate. Statistics were calculated using the ordinary two-way ANOVA with Šídák’s multiple comparisons. ns = not significant. SD, standard deviation.