Cell viability test. Cell viability was assessed by cell counting and a colorimetric assay. Cell counting was conducted in 24-well plates after seeding with 1 × 104 cells. After 1 day and 3 days of incubation with IMQ,
SB 203580 were harvested and viable cells were counted, which could be discriminated from dead cells by Trypan blue staining and colorimetric assays. The counting results are expressed as the means ± SE of triplicate measurements. The colorimetric assay was conducted using a cell viability and cytotoxicity assay kit obtained from Dojindo Molecular Technologies (Rockville, MD). In brief, Caco-2 cells were seeded at 5 × 103 cells in 96-well plates and allowed to attach to the plates for 2 days, at which time IMQ, LOX, or TNF-alpha were added. After 3 days of incubation, 10 μl of color reagent was added to each well, and the
cells were incubated for 1 h. In addition, cell suspensions made for the counting assay were distributed onto 96-well plates as 100 μl aliquots and the cells were incubated for 1 h with 10 μl of color reagent. The mean absorbance at 450 nm was measured in each set of samples. Absorbance at 450 nm was measured in each set of samples. Shown are the mean percentages of triplicate measurements ± SE, subtracted from the surviving controls, from three independent experiments. The effects of autophagy regulators on cell survival were evaluated following 1 h pre-incubations with the reagents, 3-MA (5 mM), BFA (50 nM), and rapamycin (100 nM), followed by IMQ treatment.
