Figure 2.

Methotrexate induced growth arrest in U937 monocytes is inhibited by the anti-oxidants glutathione and N-acetylcysteine. U937 monocytes (2×10⁶ ml⁻¹) were serum starved for 4 h in RMPI 1640 in the presence or absence of 10 mM N-acetylcysteine (NAC) or 10 mM glutathione (GSH) prior to the addition of 0–100 μM methotrexate for 16 h. Incubations were 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 as described in Methods. The percentage G0/G1 DNA content of 20,000 nucleoids from each sample was analysed using MultiCycle™ for Windows (Phoenix Flow Systems, San Diego, U.S.A.). The data is expressed as the mean±s.e.mean of at least four individual experiments where **(P<0.01) were considered significantly different from samples with no pre-treatment by Student's t-test. NS=no significant difference by one-way ANOVA followed by Tukey's post hoc test.