Clonogenic Survival Assay-

Clonogenic survival assays were performed as previously described.[19] Briefly, cells were plated and allowed to attach for 24 – 48 h. Cells were treated with P-AscH^- (1 and 2 mM or 10 and 20 pmol cell⁻¹, respectively) for 1 h in DMEM/10% FBS followed by irradiation at 0.5 – 2 Gy. Cells were immediately trypsinized, counted and seeded into 6-well plates at varying densities. FHs 74 Int and H6c7 cells were seeded on top of feeder cells that had been irradiated at 30 Gy. The dishes were maintained in a 37 °C, 4% O₂, for 10–14 days to allow colony formation. The colonies were then fixed with 70% ethanol, stained with Coomassie™ Blue and counted (colonies containing > 50 cells were scored). Plating efficiency was determined by the formula: (number of colonies formed/number of cells inoculated) ×100.