Nadp nadph glotm

Totally, 5 × 10³ cells were seeded per well in a 96-well plate overnight. Totally, 50 µl of NADP/NADPH-Glo^TM (Promega) detection reagent was added into each well that had the cells in 50 µl of media. The reductase in the detection reagent converted the proluciferin reductase substrate to luciferin in the presence of NADPH molecules. The plate was incubated for 60 min at room temperature and the luminescent signal, which was proportional to the NADPH amount, was measured by Tecan luminometer.

For the ATP assay, BacTiter-Glo^TM (Promega, Fitchburg, United States) was used. The culture broth was harvested in the early exponential phase (OD_600nm ≈ 1.0). First, 100 μL of harvested culture broth was mixed with an equal volume of BacTiter-Glo^TM reagent. The mixed samples were incubated at room temperature for 5 min. Luminescence was measured using a microplate reader (Synergy H1). The luminescence unit was converted to μM using a standard curve, and the result was divided by dry cell weight. Therefore, the ATP concentration was calculated as μmol/g_cell.
For the NADPH/NADP⁺ assay, NADP/NADPH-Glo^TM (Promega, Fitchburg, United States) was used. Harvested cell of early exponential phase in 50 μL of PBS was lysed in 50 μL of 0.2 M NaOH and 1% (w/v) dodecyl trimethyl ammonium bromide (DTAB). Then, 25 μL of 0.4 M HCl was treated at 60°C for 15 min. Next, 25 μL of 0.5 M Trizma base was added for neutralization, and NADP/NADPH-Glo^TM reagents were also added. Samples were incubated at room temperature for 30 min. Luminescence was measured using a microplate reader (Synergy H1). PBS, NaOH, DTAB, HCl, and Trizma base were from Sigma-Aldrich (St. Louis, MO, United States).