Rapamycin is not toxic to KG1a cells. (A, B) KG1a cells were suspended/re-suspended at 2 × 105/ml on days 0,2,4,7 and 9 of an 11 day culture in medium with 0.5% foetal calf serum, 10% foetal calf serum or 10% foetal calf serum + 100 nM rapamycin. (A) Cell growth was measured by haemocytometer; (B) apoptosis was measured by Annexin V labelling. Annexin V was measured only at 2 and 4 days on cells grown in 0.5% FCS as there were insufficient cells remaining by 7 days. (C) Following 48 hours’ culture in RPMI with 10% foetal calf serum with or without 100 nM rapamycin, cells were harvested, and processed for immunohistochemical analysis of γH2A.X foci. Cells treated for 2 hrs with 100 μg/ml etoposide were used as positive control in each assay. (For interpretation, an H score (reference 22) is a staining intensity score of 1–5 per cell for 100 cells, such that a score of 100 represents no staining and a score of 500 represents 100 completely damaged cells.). In each experiment datapoints indicate mean and standard deviation of 3 independent assays.