Figure 3.

Methotrexate mediated induction of apoptosis in Jurkat T-cells is inhibited by the anti-oxidants glutathione and N-acetylcysteine. Jurkat T-cells (2×10⁶ ml⁻¹) were serum starved for 4 h prior to the addition of methotrexate (0–1×10⁻⁴ M) for 6 or 16 h in the presence or absence of 10 mM of the anti-oxidants N-acetylcysteine (NAC) or 10 mM glutathione (GSH). Incubations were performed at 37°C in a 95% air, 5% CO₂ humidified atmosphere and terminated by washing the cells twice with ice cold PBS. Cell pellets were resuspended in 1 ml of hypotonic fluorochrome solution and incubated in the dark at 4°C overnight prior to DNA cell cycle analysis by flow cytometry. The sub-diploid DNA content of 20,000 nucleoids from each sample was analysed. The data is expressed as the mean±s.e.mean of at least four individual experiments, expressed as the percentage specific apoptosis where * represents significant difference from control samples (P<0.05) by one-way ANOVA followed by Dunnett's multiple comparison test and + represents significant difference of 16 h methotrexate samples pre-treated with NAC or GSH compared to none pre-treated samples by Student's t-test (P<0.05).