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Title: Determination of NAD + and NADH level in a Single Cell Under H 2O 2 Stress by Capillary Electrophoresis

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 H 2O 2, 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:
Research Org.:
Ames Lab., Ames, IA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
939381
Report Number(s):
IS-T 2782
TRN: US200823%%109
DOE Contract Number:  
AC02-07CH11358
Resource Type:
Thesis/Dissertation
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; ELECTROPHORESIS; SENSITIVITY; SPECIFICITY

Citation Formats

Xi, Wenjun. Determination of NAD+ and NADH level in a Single Cell Under H2O2 Stress by Capillary Electrophoresis. United States: N. p., 2008. Web. doi:10.2172/939381.
Xi, Wenjun. Determination of NAD+ and NADH level in a Single Cell Under H2O2 Stress by Capillary Electrophoresis. United States. doi:10.2172/939381.
Xi, Wenjun. Tue . "Determination of NAD+ and NADH level in a Single Cell Under H2O2 Stress by Capillary Electrophoresis". United States. doi:10.2172/939381. https://www.osti.gov/servlets/purl/939381.
@article{osti_939381,
title = {Determination of NAD+ and NADH level in a Single Cell Under H2O2 Stress by Capillary Electrophoresis},
author = {Xi, Wenjun},
abstractNote = {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.},
doi = {10.2172/939381},
journal = {},
number = ,
volume = ,
place = {United States},
year = {2008},
month = {1}
}

Thesis/Dissertation:
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