Purification of B lymphocytes and proliferation after UV-irradiation. (A) Flow cytometric analysis of naive primary B lymphocytes stained with anti-CD19 PE and anti-IgD FITC antibodies to assess purity (left) and with propidium iodide for cell cycle profile (right). (B) Differential staining of DNA with Hoechst 33342 and RNA with Pyronin Y in naive primary B lymphocytes, wild-type (WT), and XP-E lymphoblasts. The percentage of cells in G₀, G₁, and G₂ is indicated for each cell type. (C) Cell division analyses of CFSE-labeled primary B lymphocytes, WT, and XP-E lymphoblasts, after UV irradiation. Quiescent B lymphocytes were irradiated or not with 5 J/m² 254 nm UV light, pulse-labeled with the protein dye CFSE 24 hours later, and stimulated to proliferate with antihuman F(ab′)₂ fragments and Pam3CSK4 lipopeptide (IP3). WT and XP-E lymphoblasts were similarly labeled with CFSE 24 hours after irradiation. Samples were analyzed 24 or 72 hours after labeling for the dilution of the CFSE signal resulting from cell division, excluding dead cells on the basis of propidium iodide staining. The shaded peak (CTRL) corresponds to the autofluorescence of unlabeled cells. Data are representative of 3 independent experiments. (D) Time course of [³H]thymidine incorporation in cells irradiated or not with 5 J/m² UV light. After irradiation, quiescent B lymphocytes were cultured for 24 hours before stimulation. In all experiments, [³H]thymidine was added 20 hours before sample collection. Data are displayed as means of counts per minute (cpm) ± SEM of 2 independent experiments.