Nadp nadph assay kit

NADPH levels were analyzed using an NADP/NADPH Assay Kit (ab65349, Abcam). Cells were seeded in 10 cm² plates and treated with DMSO, erastin, or RSL3 for 12 h. Cells were lysed in a 200 μL NADP/NADPH extraction buffer. The samples were then briefly vortexed and centrifuged at 14,000 g for 5 min. The supernatants were passed through spin columns provided in the kit and centrifuged at 10,000 g for 40 min. The filtered samples were then heat-treated at 60 °C for 30 min to decompose NADP. Fifty microliter standards or samples were added into the individual wells of a 96-well plate, which had preadded 100 μL reaction mix (98 μL NADP cycling buffer and 2 μL NADP cycling enzyme mix). Then, 10 μL NADPH developer (ab65349, Abcam) was added to each well, and the reaction extended for 2 h. After the reaction period, colorimetric reading was measured spectrophotometrically at 450 nm. The concentrations of these metabolites were normalized to the amount of total protein.

The NADP/NADPH ratio was measured using a NADP/NADPH assay kit (Abcam, ab65349, Cambridge, UK) according to the manufacturer's instructions. Briefly, cells harvested from a 10 cm dish were immediately lysed in 400 μL of extraction buffer, followed by two freeze/thaw cycles (20 min on dry ice followed by 10 min at RT). The lysate was centrifuged at 13 000 × g for 5 min. Two‐hundred mictroliters of supernatant from each sample were heated at 60 °C for 30 min in a water bath and then measured by reaction with the reaction mixure. The optimal readouts were collected after 1 h at room temperature.

Following 24 hours of co-culture with infected and treated macrophages, Caco-2 cells were lysed, and their levels of NADPH and total NADP were measured using the NADP/NADPH Assay Kit (Abcam, Cambridge, UK) according to manufacturer protocols. Briefly, the cell lysates were halved, with one half heated for 30 min to degrade the oxidized NADP+ while leaving the NADPH untouched. Each lysate was then mixed with the kit developer in triplicate on a 96 well plate and left to incubate in the dark for 24 hours. NADP was then quantified for each well, with the heated lysate measuring the reduced NADPH as a fraction of total NADP.