Enzychrom nadp nadph assay kit

The cellular NADPH production was determined using the EnzyChrom^TM NADP⁺/NADPH assay kit (Bioassay Systems, Hayward, CA, USA), as previously described (10 (link)). The protein concentration of the samples was determined using protein quantification, as previously described (17 (link)). The NADPH concentration was normalized to the total protein, and presented as μM/min/mg total protein. In total, at least three independent experiments were performed.

NADPH levels were determined by using EnzyChrom™ NADP⁺/NADPH assay kit (BioAssay Systems, Hayward, CA, USA) following the manufacturer’s instructions.

We extracted the total protein from mouse liver tissue lysate and quantified it by Bio‐Rad protein assay reagent as per the previous study.²⁵ The liver NADPH oxidase was detected by EnzyChrom NADP/NADPH assay kit (BioAssay Systems), as per the manufacturer's instructions.

Measurement of NADPH concentration was based on a glucose dehydrogenase cycling reaction. The level of NADPH in kidney mitochondria was determined using an EnzyChrom™ NADP/NADPH Assay Kit (BioAssay Systems, Hayward, CA, USA) according to the manufacturer’s instruction. In brief, freshly isolated kidney mitochondria was suspended in 100 μL NADPH extraction buffer for NADP determination or 100 μL NADPH extraction buffer for NADPH determination, and then heated at 60 °C for 5 min. After heating 20 μL assay buffer and 100 μL of the NADP, opposite extraction buffer was added to neutralize the extracts. After centrifugation of the sample at 14,000 r.p.m. for 5 min, the supernatant was used for determination. In 96-well plate, 40 μL samples were transferred and added 80 μL working reagent (60 μL assay buffer, 1 μL enzyme mix, 10 μL glucose, and 14 μL MTT). The plate was briefly tapped to mix and read the optical density (OD) at 565 nm at time zero and 30 min. The concentration of NADP(H) was calculated using the change of OD for 30 min.

The lungs were homogenized in NADP or NADPH extraction buffer. The NADP⁺ and NADPH in the protein extracts were measured using the EnzyChrom NADP⁺/NADPH assay kit (BioAssay Systems, Hayward, CA) based on a glucose dehydrogenase cycling reaction, in which the formed NADPH reduces a formazan (MTT) reagent. Each sample was run in duplicate and expressed as NADP⁺/NADPH ratio according to the manufacturer’s instructions.

The concentration of NADPH was measured by EnzyChrom™ NADP⁺/NADPH assay kit purchased from BioAssay Systems (Hayward, United States). 1×10⁴ BV2 cells were seeded in 24-well plates, and treated with LPS (100 ng/ml) with or without RRx-001 (5 μM). After 24 h, the cells were collected and the samples were prepared according to the manufacturer’s instructions. The absorbance was detected at 565 nm by using infinite M1000 PRO multiscan spectrum (Tecan, Switzerland).