Assessment of NADPH/NADP Ratio in Primary Microglia

Nicotinamide nucleotides were assayed using the NADP/NADPH assay kit according to the manufacturer’s instructions. Primary microglia were seeded onto PDL-coated 12-well plates and treated with the given concentrations of LPA for the indicated time periods. NADP and NADPH were extracted using the provided extraction buffer.

The samples need to be deproteinized using 10 kDa Spin Columns (Abcam, Cambridge,
UK) before performing the assay, yet the columns are not provided along with the kit and they need to be bought separately.

An aliquot of the sample was used to measure total NADPt (NADP and NADPH). Another aliquot of the sample was heated at 60°C for 30 min to decompose NADP for NADPH measurement. 10 µL of the sample were mixed with NADP cycling mix to convert NADP to NADPH. Thereafter, 10 µL of NADPH developer were added into each well, mixed, and incubated at room temperature for 1–4 h.

The ratio of NADPH/NADP was calculated as follows: NADPH/NADP ratio = NADPH/(NADPt - NADPH).

The procedure is relatively fast and the kit worked as expected. However, a certain degree of variation was observed between individual experiments and it is strongly recommended to repeat the experiment at least 4 times in triplicate.