Cell Culture Conditions for KSHV and EBV Studies

The KSHV-positive and EBV-negative PEL cell lines BC-3 and BCBL-1, and KSHV-negative and EBV-negative B lymphocyte lines DG75, Loukes and BJAB cells were maintained in RPMI-1640 containing 10% heat-inactivated fetal bovine serum (FBS), 2 mmol/L of L-glutamine, 100 U/ml of penicillin, and 100 μg/mL of streptomycin at 37°C in a humidified, 5% CO₂ atmosphere. HEK293T and EA.hy926 cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS. EA.hy926 is an immortalized cell line obtained from fusion of primary human umbilical vein cells and the A549 human lung adenocarcinoma cell line, which has the characteristics of vascular endothelial cells [64 (link)]. Primary human umbilical vein endothelial cells (HUVECs) were isolated from the interior of the umbilical vein of human umbilical cords by digestion with collagenase (Sigma, St. Louis, MO, USA) as described [65 (link)]. HUVECs were cultured in complete EBM-2 culture media (LONZA, Allendale, NJ, USA) and used between passage 3 and 6. Wild type recombinant KSHV BAC16 and a KSHV mutant with miR-K3 deleted, BAC16ΔmiR-K3, were previously described [39 (link), 66 (link)].