Nad nadh glotm assay kit

To measure the intracellular NADH/NAD⁺ ratio, NAD/NADH‐GloTM Assay kit (Promega, WI, US) was utilized. Briefly, the cell broth was prepared by cultivating to exponential phase using production medium. The culture broth was mixed with DTAB solution for 5 min. For measuring NADH, 0.4 N of HCl was added to the reaction mix, whereas for measuring NAD⁺, nothing was added. The sample was heated to 60°C for 15 min and cooled to 25°C. The HCl/Trizma solution was added to the reaction mix for NADH, whereas Trizma solution was added to that for NAD⁺. The prepared solutions were mixed with detection reagent at a ratio of 1:1 (v/v) and incubated for approximately 30 min. The luminescence was measured using microplate reader Biotek Synergy H1 (Biotek, VT, USA). The same methods were applied for measuring NAD(H) standard solution. The NADH and NAD⁺ were quantified, and the ratio of them was calculated.
For measuring the intracellular ATP content, BacTiter‐GloTM kit was utilized (Promega, WI, USA). Briefly, the cell broth was prepared by flask culture. The cell broth in exponential phase was taken and washed using distilled water. The same volume of cell broth and reaction mix was resuspended together and rested for 5 min in ambient condition. The luminescence was measured using microplate reader Biotek Synergy H1 (Biotek). The same methods were applied for measuring the ATP standard solution.

To analyze the intracellular NADP⁺/NADPH ratio, E. coli were inoculated and cultured in LB medium for 16 h to reach stationary phase, and the cells were harvested by centrifugation (3300×g, 4 °C, 10 min). Cells were then washed with ice-cold phosphate-buffered saline, lysed with NADP⁺/NADPH extraction buffer (Biovision, Milpitas, CA, USA) in a microcentrifuge tube, and kept on ice for 10 min. The crude extracts were centrifuged at 15,000×g for 10 min to obtain the supernatant. The amount of NADP⁺/NADPH in the cells was measured using a NADP⁺/NADPH Quantitation Colorimetric Kit (Biovision). To measure the intracellular NADH/NAD⁺ ratio, a NAD/NADH-GloTM Assay Kit (Promega, WI, USA) was used. The culture medium was mixed with a DTAB solution for 5 min. To measure NADH, 0.4 N HCl was added to the reaction mixture. Samples were heated to 60 °C for 15 min and cooled to 25 °C before assay, following the manufacturer’s instructions.

GSH and GSSG concentrations were measured with the GSH/GSSG-Glo^TM assay kit (Promega V6612, Madison, WI). NADP⁺/NADPH levels were quantified using the NADP/NADPH-Glo™ Assay kit (Promega G9082, Madison, WI). NAD⁺/NADH levels were measured with the NAD/NADH-Glo^TM assay kit (Promega G0972, Madison, WI). ATP levels were measured with the Cell Titer-Glo Luminescence Cell Viability Assay Kit (Promega, Madison, WI)^{22 (link)}.