Determination of NAD+ and NADH level in a Single Cell Under H2O2 Stress by Capillary Electrophoresis, (Thesis/Dissertation).

Xi, W.

Technical Information Center Oak Ridge Tennessee

关键词: Electrophoresis;    Sensitivity;    Capillaries;    Fluorescence;    Specificity;   

RP-ID  :  DE2008939381

学科分类：工程和技术（综合）

美国|英语

来源: National Technical Reports Library

【 摘 要 】

A capillary electrophoresis (CE) method is developed to determine both NAD(sup +) and NADH levels in a single cell, based on an enzymatic cycling reaction. The detection limit can reach down to 0.2 amol NAD(sup +) 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(sup +) and NADH were separated, incubated with the cycling buffer, and quantified by the amount of fluorescent product generated. NADH and NAD(sup +) levels of single cells of three cell lines and primary astrocyte culture were determined using this method. Comparing cellular NAD(sup +) and NADH levels with and without exposure to oxidative stress induced by H(sub 2)O(sub 2), it was found that H9c2 cells respond to the stress by reducing both cellular NAD(sup +) and NADH levels, while astrocytes respond by increasing cellular NADH/NAD(sup +) ratio.