Gsh colorimetric detection kit

1 × 10⁶ BRx68 cells were cultured with or without 300 μM NAC for 6 or 13 days. At the end of incubation periods, cells were washed in ice cold PBS, lysed in 5% 5-sulfo-salicylic acid dehydrate by vortexing and repeating cycles of freeze-thaw, then centrifuged at 14000 rpm for 10 min and supernatant was transferred to clean tubes. GSH was measured using a GSH colorimetric detection kit (Invitrogen) following manufacturer instructions.

The Ferric Reducing Antioxidant Power (FRAP) was measured using the Ferric Antioxidant Status Detection Kit (Invitrogen). Cells were lysed and prepared according to the kit instructions. Antioxidant power was determined by comparison against a Ferrous Chloride standard provided in the kit. Absorbance was read at 506 nm on a Spectra MAX Plus scanning multi-well spectrophotometer (Molecular Devices). A minimum of three replicates were analyzed to calculate the means and standard deviations. The total intracellular glutathione content was measured using the GSH Colorimetric Detection Kit (Invitrogen). Cells were lysed and resuspended according to kit specifications. An oxidized glutathione standard was used to prepare a calibration curve. The absorbance was measured at 405 nm on a microplate spectrophotometer. A minimum of three replicates were analyzed to calculate the means and standard deviations.