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Title: 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+ 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.
Authors:
 [1]
  1. Iowa State Univ., Ames, IA (United States)
Publication Date:
OSTI Identifier:
939381
Report Number(s):
IS--T 2782
TRN: US200823%%109
DOE Contract Number:
AC02-07CH11358
Resource Type:
Thesis/Dissertation
Research Org:
Ames Lab., Ames, IA (United States)
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