Fastprep 24 classic benchtop homogenizer

The intracellular concentration of NADPH was determined using the NADP/NADPH Assay Kit (Abcam, Cambridge, MA, USA). Mid-log phase cultures (OD600 0.4–0.45) of TIGR4 and ΔpotABCD grown in THY (n = 33) were harvested at 5000× g for 10 min at 4 °C, suspended in PBS and transferred to beadbeater tubes (MP Biomedicals, Irvine, CA, USA). Cell suspensions were lysed with a FastPrep-24 ™ Classic benchtop homogenizer (MP Biomedicals, Irvine, CA, USA) and centrifuged at 6000× g for 5 min at 4 °C. The cells were processed according to the manufacturer’s instructions. NADPH concentrations were determined with a SpectraMax ^® M5 multi-mode microplate reader (Molecular Devices, Sunnyvale, CA, USA). The concentration of the protein extracts was determined with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and used to normalize NADPH concentrations.

The ratio of reduced and oxidized intracellular glutathione concentrations in TIGR4 and ΔspeA were determined using the GSH/GSSG-Glo™ Assay Kit (Promega, Madison, WI, USA). Mid-log 10 mL cultures, n = 3 were harvested at 5000× g for 10 min at 4 °C, suspended in PBS, and transferred to beadbeater tubes (MP Biomedicals, Irvine, CA, USA). Cell suspensions were lysed with a FastPrep-24™ Classic benchtop homogenizer (45 s, 6.5 m/s × 3) (MP Biomedicals, Irvine, CA, USA) and clarified by centrifugation at 6000× g for 5 min at 4 °C. The extracts were then processed according to the manufacturer’s instructions. Luminescence was measured with a Cytation™ 5 cell imaging multi-mode reader (BioTek, Winooski, VT, USA) and used to calculate glutathione concentrations. Protein concentrations of the extracts were determined with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and used to normalize glutathione concentrations. GSH/GSSG (reduced/oxidized glutathione) ratios were calculated from the normalized glutathione concentrations according to the kit manufacturer’s instructions.