Nadp nadph assay kit

After 9 h of attachment, ASCs were collected for the following assays according to the manufacturer's instructions. G6PDH (glucose-6-phosphate dehydrogenase) activity was examined in cell lysates using G6PDH Activity Assay Kit (S0189, Beyotime) and normalized to total protein contents. NADPH and NADP⁺ was extracted and measured using NADPH/NADP⁺ Assay Kit (S0179, Beyotime), the amounts were normalized to total protein contents and NADPH/NADP⁺ ratio was calculated.

Intracellular NADPH/NADP⁺ levels were assayed using NADPH/NADP⁺ assay kit (Beyotime, Shanghai, China) according to the manufacturer's instructions. Briefly, 2 × 10⁶ cells were washed with cold PBS and pelleted. Homogenized samples were treated with 200 μL counter NADPH (or NADP⁺) extraction buffer, which were incubated at 60 °C for 5 min. The 20 μL of assay buffer and 200 μL of the counter NADPH (or NADP⁺) extraction buffer were added to neutralize the extracts. Then, these extracts were centrifuged at 12,000 rpm for 5 min on a Legend Micro 21R Centrifuge (Thermo Fisher Scientific, MA, USA), and the NADP⁺ or NADPH was measured in the supernatant by reading the fluorescence at 450 nm according to the manufacturer's protocol on a microplate photometer. NADPH/NADP⁺ ratios were calculated according to the manufacturer's protocol.