Infinite m100 pro microplate reader

Cellular glutathione was assayed using a GSH-Glo kit (Promega) as outlined by the manufacturer. Cells (20,000) were seeded in triplicate in a white clear-bottom 96-well plate. The following day, they were treated with MMS (0.15 or 1 mM) or H₂O₂ (350 or 500 μM) for 1 h. In some studies, cellular glutathione was significantly depleted by treatment of cells for 16 h with 15 μM BSO. Cells were then washed, and incubated with control medium for 3 h. A 1x solution of GSH-Glo Reagent was prepared by adding 100 μl of Luciferin-NT substrate, 100 μl of glutathione-S-transferase, and 100 μl of 100 mM TCEP (final concentration of 1 mM) to 10 ml of GSH-Glo Reaction Buffer. Medium was removed from the cells and GSH-Glo reagent (100 μl) was added to each well and incubated at room temp for 30 min. Reconstituted Luciferin Detection Reagent (100 μl) was then added to each well and incubated at room temperature for 15 min. Luminescence was measured using a Tecan Infinite M100 Pro microplate reader. To determine background levels, 10 μl of H₂O was added to an empty well. A standard curve was used to calculate total glutathione, subtracting out the background reading from all sample readings.

17β-estradiol (E2) levels in cell culture media were determined using ELISA Kit from Cayman Chemical (Ann Arbor, MI), according to manufacturer’s protocol [17 (link)], optimized further for better efficacy. Briefly, culture media collected from various cell lines and/or treatment groups were extracted with diethyl ether (5:1, v/v), snap froze samples in ice/ethanol bath, and poured top solvent layer into another tube. Solvent samples containing E2 were dried with air or in a speedvac, resuspended in assay buffer, and E2 levels were measured. The sensitivity of E2 assay was 15pg/ml, and intra-assay percentage coefficient of variation was below 10%. Assays were performed at duplicates and absorbance was read at 412nm using an Infinite M100 PRO Microplate Reader (Tecan, Mannedorf, Switzerland).