Sulindac

CL1-1, CL1-5, or A549 cells (1x10⁴) were seeded for 24 h at 37°C in a 96-well culture plate, then were subjected to starvation for 14 h in RPMI 1640 medium containing 2% fetal calf serum, 100 Units/ml of penicillin, and 100 mg/ml of streptomycin. Following 6 h pretreatment with medium or the indicated concentration of sulindac or its metabolites (all from Sigma), the cells were incubated for 12 h with or without the indicated concentration of β-lapachone in the continued presence of sulindac or its metabolites, and then cell viability was evaluated.
Two cell viability assays were used. In the crystal violet staining assay, the cells were fixed with 4% paraformaldehyde for 15 min, stained with 0.4% crystal violet for 15 min, and washed with H₂O, then 50% acid alcohol was used to dissolve the bound crystal violet and the OD at 550 nm measured on an ELISA reader. In the MTT assay, 10 µl of MTT (0.5 mg/ml) (Sigma) was added to each well and the plates incubated at 37°C for 4 h, then the formazan product was dissolved in 100 µl of DMSO at 37°C for 30 min and the OD at 570 nm measured on a microplate reader.

BxPC3 cells were grown in RPMI medium. T84 cells were grown in 1:1 DMEM/F12 medium. For sulindac treatment, cells were incubated with 400 μM sulindac (Sigma) for 48 h before harvesting. Controls were treated with DMSO, the NSAID vehicle.