SciTech Connect

Title: Determination of NAD+ and NADH level in a Single Cell Under H2O2 Stress by Capillary Electrophoresis

Determination of NAD+ and NADH level in a Single Cell Under H2O2 Stress by Capillary Electrophoresis 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.
Authors:
Publication Date:
OSTI Identifier:OSTI ID: 939381
Report Number(s):IS-T 2782
TRN: US200823%%109
DOE Contract Number:DE-AC02-07CH11358
Resource Type:Thesis/Dissertation
Research Org:Ames Laboratory (AMES), Ames, IA
Sponsoring Org:USDOE Office of Science (SC)
Country of Publication:United States
Language:English
Subject: 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; ELECTROPHORESIS; SENSITIVITY; SPECIFICITY