Abstract
A capillary electrophoresis (CE) method is developed to determine both NAD and NADH levels in a single cell, based on an enzymatic cycling reaction. The detection limit can reach down to 0.2 amol NAD and 1 amol NADH on a home-made CE-LIF setup. The method showed good reproducibility and specificity. After an intact cell was injected into the inlet of a capillary and lysed using a Tesla coil, intracellular NAD and NADH were separated, incubated with the cycling buffer, and quantified by the amount of fluorescent product generated. NADH and NAD levels of single cells of three cell lines and primary astrocyte culture were determined using this method. Comparing cellular NAD and NADH levels with and without exposure to oxidative stress induced by H2O2, it was found that H9c2 cells respond to the stress by reducing both cellular NAD and NADH levels, while astrocytes respond by increasing cellular NADH/NAD ratio.
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