2.7. Intracellular NADP+/NADPH ratio measurements

The intracellular NADPH level and NADP+/NADPH ratio were determined using the Calorimetric NADP+/NADPH Assay Kit (Cell Biolabs), according to the manufacturer’s protocol. Harvested cells were resuspended at 2 × 10⁶ cells/mL in extraction buffer and homogenized on ice. The supernatant was filtered with a 10 kDa spin filter for deproteination and total NADP+/NADPH quantification was immediately carried out. Extraction of NADPH and NADP+ included the addition of NaOH and HCl to eliminate NADP+ and NADPH, respectively, followed by incubation at 80°C for 1 hour and pH neutralization. Samples were then mixed with NADP Cycling Reagent and incubated for 2 hours at room temperature protected from light. The NADP+/NADPH concentrations were calculated by comparing the sample optical density read with a Multiskan GO spectrophotometric microplate reader at 450 nm.