Quantitative and qualitative comparison of different EBV transformation methods. A) The B-lymphocyte subsets were prepared using the SEQUENTIAL, COMBINED or BASIC methods, as outlined in Fig. 1. Ten days after exposure to EBV, samples of each population were compared for the total cell number (black bars), and for the number of viable cells (white bars) measured microscopically by trypan blue dye exclusion. Data are expressed as % of initial B cells exposed to EBV and represent the means ± s.d. of 5 cell counts for each condition. Results are representative of two independent experiments. P < 0.001: SEQUENTIAL vs. COMBINED; BASIC vs. COMBINED (white bars); P < 0.05: SEQUENTIAL vs. BASIC (white bars) and SEQUENTIAL vs. COMBINED; and BASIC vs. COMBINED (black bars). B) Ten days after infection with EBV, the cells prepared by the SEQUENTIAL, COMBINED, or BASIC method were analyzed to identify viable lymphoblasts by propodium iodide (PI) exclusion (top panels) and CD23 expression (bottom panels) by flow cytometry. For each population of cells, the viable lymphoblasts (gated in regions R2, top panels) were defined as those with relatively high forward scatter (horizontal axes) and negative for PI fluorescence (vertical axes). R2 gated cells were then analyzed for CD23 expression. Fluorescence was analyzed with a FACSCalibur flow cytometer and CellQuest software (Becton Dickinson). Background fluorescence was determined with a FITC-labeled, isotype-matched negative control mAb. 10,000 events were measured for each condition.