Analysis of cell growth

At the indicated time points, cells were collected and resuspended in 100 µl FACS buffer and analyzed by flow cytometry in the presence of 1 µl CountBright Absolute counting beads for flow cytometry (Invitrogen, Cat. No. {"type":"entrez-nucleotide","attrs":{"text":"C36950","term_id":"2373091","term_text":"C36950"}}C36950) until 400 to 500 beads were acquired (as gated by size and fluorescence intensity). The concentration of cells was determined as described by the manufacturer. From this value, the total cell number for each culture was calculated based on the total volume of the cell culture and the recorded split ratio (since day 3). Cells were split at a ratio of up to 1:2, when necessary, as judged by cell density and medium pH, and only when they reached more than 1 million cells/ml (resulting in newly split cells at a concentration of 1-2 million/ml). Cells were pipetted up and down softly to break up larger cell clumps. Between day 25 and 37 (the time window when LMP1 single-positive cells ceased to grow), cultures were not, or only rarely, split. Cell viability was measured by trypan blue dye exclusion in an automated cell counter (TC20, BIO-RAD) with particles with a diameter below 5 μm excluded.