Medium thiol quantification

Cells were washed once with phenol red free growth media and then 10,000 cells were plated with the 100 μl of phenol red free growth media in a 96 well plate. 100 μl of conditioned media were collected and directly mixed with 50 μl of 10 mM DTNB (5,5’-dithiobis-(2-nitrobenzoic acid)) dissolved in DMSO after 24 hr conditioning. Absorbance at 450 nm was measured spectrophotometrically in 5 min (DTX880, Beckman Coulter). The remaining cells in the same plate were subjected to CellTiter-Glo assay for normalization. Absorbance value of the blank was subtracted as background, and values were normalized total viable cells as determined by CellTiterGlo. Due to the masking effect of phenol red on DTNB absorbance, created by reacting with thiols, phenol red free media was used for all thiol quantifications.