Nadph glo kit

Bovine serum albumin (BSA), NADPH, human glutathione reductase (hGR), oxidized glutathione (GSSG) and 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB), IMDM medium (Iscove’s Modified Dulbecco’s Medium), sodium bicarbonate, hypoxanthine, thymidine, bathocuprione sulfonic acid, cysteine, β-mercaptoethanol, heat-inactivated Calf serum, triton-X 100, anti-His tag antibody and protease inhibitors cocktail were purchased from Sigma-Aldrich (St. Louis, USA); oxidized trypanothione (TS₂) was purchased from Bachem (Bubendorf, Switzerland); NADPH-Glo kit and CellTiter-Glo were purchased from Promega (Madison, WI).

Competition assays were performed using three test concentrations (4, 16 and 64 μM) for the competing compounds. The reaction buffer and components were mostly identical to those used in the TR assay described above, but 1 nM TR was employed for 5 minutes against a serial dilution of either TS₂ in presence of 40 μM NADPH or NADPH in presence of 30 μM TS₂. The residual amount of NADPH was measured using the NADPH-Glo kit (Promega, U.S.A.) after 30 min incubation at room temperature as per the manufacturer’s protocol. The luminescent signal was measured using the EnVision plate reader (PerkinElmer, USA). IC₅₀, V_max, and K_m values were calculated using Prism software (GraphPad, San Diego, CA, U.S.A.).