Figure 5. Efficient Proliferation and Survival of EBV-infected B cells require LMP2.

1×10⁶ purified B cells were labeled with Violet Tracer (Invitrogen) and infected with recombinant wt and LMP2 KO EBV at MOI 1. B cells were harvested at 4, 7 and 14 days post-infection and stained for Annexin V. During fixation step, 10 μl counting beads (CountBright Absolute Counting Beads, Invitrogen) were added to each sample. During acquisition, the event gate was set to 5000 beads, which normalized the acquisition volume between samples and allowed for accurate, absolute counts of proliferating B cells. (A) Gating strategy for proliferation and apoptosis analysis. Doublets and dead cells were excluded. Next, lymphocytes and EGFP+ cells were selected for proliferation and apoptosis analysis. (B) Representative donor for proliferation and apoptosis data. (C) Proliferation and (D) Apoptosis data points are an average of 3 independent experiments ± SEM. Statistical significance was determined using a Two-Way ANOVA test and Bonferroni Post-test (each data point for LMP2 KO viruses was compared to wild-type infection to determine statistical significance). P-value (*) <0.05, p-value (**) <0.01 and p-value (****) <0.0001.