2.4. NAD/NADH Assay

The NAD⁺/NADH ratio was measured using an NAD⁺/NADH assay kit (Abcam, Waltham, MA, USA). The detailed experiments were performed according to the procedures provided by the kit. S. aureus treated with or without gA and cations incubated at 0, 1, 2, and 3 h were harvested and centrifugated at 10,000× g for 15 min at 4 °C. The cells were resuspended in a lysis buffer to a final concentration of 10⁷–10⁸ cells/mL. The resuspended cells were incubated at room temperature for 15 min and centrifugated at 2500 rpm for 5 min. The supernatant was transferred to a new tube and kept on ice.

For NADH extraction, twenty-five μL of the sample with or without gA and cations was added to a well in a 96-well plate. Then, twenty-five μL of NADH extraction buffer was added to the well containing the sample and incubated for a further 15 min at 37 °C. After incubation, twenty-five μL of NAD⁺ extraction buffer was added to neutralize NADH extraction. Similar procedures were performed for NAD extraction, except that NAD extraction buffer was used to extract NAD, and NADH extraction buffer was used to neutralize the reaction. For the NAD⁺/NADH ratio, seventy-five μL of NADH reaction enzyme mixture was added into each well and incubated at room temperature for 2 h. The related concentration of NAD or NADH was measured using a fluorescence microplate reader (FlexStation 3, MD, San Jose, CA, USA) with an excitation wavelength of 540 nm and emission wavelength of 590 nm.