Nadph assay kit

This assay was carried out with an NADPH Assay kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. Briefly, an NADPH standard solution was prepared to quantify the NADPH content of samples. Then, 50 µL/well of cell lysates were added to a 96-well plate, mixed with 50 µL/well of NADPH reaction mixture and incubated for 2 h at room temperature. The absorbance was measured at 460 nm, and values were compared with the standard curve. The NADPH level was calculated by normalizing the measured NADPH concentration by the protein content of the cell lysate.

For studying the involvement of oxidative stress, antioxidant enzyme activity of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), glutathione reductase (GR), Reduced nicotinamide adenine dinucleotide phosphate (NADPH) and thio-barbituric acid reactive substances (TBARS) were analysed. The isolated ovary tissue (20 mg) was homogenised using a tissue homogeniser in 2 ml phosphate buffer of pH7.4 followed by centrifugation at 10,000 g for 25 min at 4 °C. The supernatant of the resultant was used for measuring all the oxidative stress markers. The CAT and GR were evaluated using Catalase assay and glutathione reductase kit respectively (Sigma Aldrich USA), POD and NADPH by peroxidase assay and NADPH assay kit respectively (Abcam USA), SOD by superoxide dismutase kit (ThermoFisher USA), TBRAS using a TBRAS assay kit (Cayman Chemical, USA). All the procedures were in accordance to supplied instructions.

The NADPH/NADP ratio was determined using a NADPH Assay Kit (ab65349, Abcam). Trypsinized cells were lysed in 800 μL of assay buffer and homogenized with two freeze/thaw cycles (20 min on dry ice followed by 10 min at room temperature). Samples were vortexed and centrifuged at top speed for 5 min. Then, samples were passed through a needle-fitted syringe to shear DNA. To detect NADPH, NADP needs to be decomposed before the reaction. Aliquots containing 200 μL of extracted samples were placed into new e-tubes and heated to 60 °C for 30 min, and 50 μL of sample was added to each well of a 96-well plate. A reaction mix containing 98 μL NADPH Cycling Buffer and 2 μL of NADPH Cycling Enzyme Mix was made, and 100 μL of the reaction mix was added to each well containing a test sample. Then, the plate was incubated at room temperature for 5 min and 10 μL NADPH Developer was added into each well. The plate was then incubated at room temperature for 1–4 h. The absorbance was measured using a microplate reader at OD 450 nm.

Cellular NADPH concentrations were measured using NADPH assay kit (Abcam). Briefly, PDAC cells were lysed with 400 μl of NADP⁺ extraction buffer and heated at 60 °C for 30 min. Twenty microliters of assay buffer and 200 μL of NADPH extraction buffer were added to neutralise the extracts. The extracts were used to determine the NADPH concentration. The absorbance of the reaction mixture at 565 nm was measured by a plate reader at 0 and 30 min.