Nadph glo assay

Cellular NADH/NAD⁺ and NADPH/NADP⁺ ratios were measured using NADH-glo assay and NADPH-glo assay respectively (Promega, G9072 and G9082) according to the manufacturer’s instructions. Briefly, 5 × 10⁴ cells were seeded in a 24-well plate. 24 h after seeding, the medium was exchanged for DMEM with doxycycline or water. At the indicated time points after doxycycline addition, the cells were quickly washed with PBS and then 0.2 mL of an ice-cold 1:1 mixture of PBS and bicarbonate base buffer with 1% DTAB was added to lyse the cells. 50 µL of the cell lysate was transferred to an empty well of 96 well plates for the base treatment. For the acid treatment, the same volume of cell lysate and 25 µL of 0.4 N HCl were added. Both samples were incubated for 15 min at 60 °C to destroy reduced or oxidized nucleotides. 25 μL of 0.5 M TrizmaR base was added to each well of acid-treated cells to neutralize the acid and 50 μL of HCl/TrizmaR solution was added to each well containing base-treated samples. The cellular NADH/NAD⁺ and NADPH/NADP⁺ ratios were determined with the kits following the manufacturer’s instructions, and the amounts of NADH, NAD⁺, NADPH, or NADP⁺ were normalized by cell numbers.

The end-point assay was developed using NADPH-Glo Assay (Promega, cat. #G9082). The linear range of the kit was established by performing serial dilution of NADP⁺. Due to the low NADP⁺ detection limit, reaction conditions were optimized to 100-pM Mtr_Mtb, 30-µM substrate and 15-µM NADPH. To establish the linear velocity phase of the primary reaction, a series of reactions were stopped every 10 min from 20 to 60 min by addition of 1-M HCl. The NADP⁺ standard curve was included in the plate setup to determine the reaction turnover rate. Following the manufacturer’s protocol, the plate was incubated for 30 min at 25°C. Next, reaction mixtures were neutralized with 1.5-M NaOH following 30-min equilibration. The plate was centrifuged for 1 min at 200 × g. The readout solution was added to the wells in a 1:1 ratio. Reaction progress was measured kinetically over 50 min on TECAN M200 (integration time 0.3 s). The time of the end-point reading was set based on the substrate turnover determined from the NADP⁺ standard curve. The quality and robustness of the assay were examined by calculation of the Z-factor, according to equation 2 (45 (link)) and signal-to-background ratio (S:B; S:B = mean signal/mean background).