Bactiter glotm 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.

The luminescent BacTiter-Glo^TM kit (Promega) was used to quantify the ATP levels in untreated and CBG-treated samples. Briefly, 100 μl of each sample (CBG = 0, 2.5, 5, 10, and 20 μg/ml for 2 h) was mixed with 100 μl of the reagent in 96-flat bottom plates (Greiner Bio-One, μClear white clear bottom plates), and after mixing for 5 min on an orbital shaker, the luminescence was recorded using the M200 Tecan plate reader. ATP level was calculated in comparison to the control using the following equation: (sample luminescence/control luminescence) × 100.