Facscalibur g 5 system

To induce lytic replication in HEK-293 cells carrying EBV-BAC, cells were transfected with pcDNABZLF1 using the Neon transfection system. After 3 days, cells and culture media were collected and subjected to centrifugation. Next, Akata(−) cells were infected with the virus solution for 3 h at room temperature with rotation. After 2 days, the cells were fixed with 1% formaldehyde, washed with PBS, and resuspended in PBS. GFP-positive cells were counted using the FACS Calibur G5 system (Becton, Dickinson), according to the manufacturer’s instructions.

To induce lytic replication in HEK293 cells carrying EBV BAC clones, cells were transfected with pcDNABZLF1 using the Neon transfection system (Invitrogen). At 72 h after transfection, cells and culture medium were collected, followed by freezing and thawing. After centrifugation at 13,000 rpm, the supernatants were mixed to Akata(-) cells for 3 h at room temperature with rotation, and the medium was replaced with fresh medium. At 48 h post-infection, cells were fixed with 1% formaldehyde, washed with PBS, and suspended in PBS. GFP-positive cells were counted using the FACSCalibur G5 system (Becton-Dickinson), according to the manufacturer’s instructions.