High Throughput Method for Assessment of Cellular Reduced Glutathione in Mammalian Cells.
Reduced glutathione (GSH) is an intracellular molecule essential for many aspects of cell physiology and defense. Determination of GSH has been used to identify potential anti-cancer drugs and for the assessment of drug toxicity via generation of oxidative stress. The described protocol was designed to modify existing protocols for the fluorescent detection of intracellular GSH in a high throughput 96-well microplate format. Dibromobimane was used to label intracellular GSH, and an additional dye, Hoechst 33342 was used to measure cell density for data normalization. Cell density curves were performed using HEK 293T cells to determine the optimal starting cell density, (< 8.0 × 10(4) cells/well) for fluorescent analysis. Fluorescent dyes were also analyzed for compatibility and spectral overlap. The method was further validated by exposing HEK 293T cells to GSH modulating agents; tert-butylhydroquinone a potent inducer of GSH, and L-buthionine-(SR)-sulfoximine a potent inhibitor of GSH. This study provides a fast, simple method for the high throughput screening of GSH in a widely available 96-well format. It also addresses the pitfalls associated with fluorescent compounds in cell culture and proper data normalization.
Cox
David P
DP
Center for Environmental Health Sciences, Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, Montana, U.S.A.
Cardozo-Pelaez
Fernando
F
Cytometry. 1994 Apr 1;15(4):349-58
8026225
J Biol Chem. 1982 Nov 25;257(22):13704-12
6128339
Annu Rev Pharmacol Toxicol. 1999;39:67-101
10331077
Glia. 2000 Jun;30(4):329-41
10797613
Toxicol Sci. 2004 Nov;82(1):308-17
15282410
J Chromatogr. 1991 Jul 17;568(1):219-25
1770099
Free Radic Biol Med. 1999 Nov;27(9-10):916-21
10569624
Cytometry A. 2003 Jan;51(1):16-25
12500301
Anal Biochem. 1969 Mar;27(3):502-22
4388022
Mutat Res. 1988 Dec;202(2):343-61
3057366
Anal Biochem. 2000 Apr 10;280(1):80-6
10805524
Cytometry. 1997 Sep 1;29(1):76-82
9298814
Biomed Chromatogr. 2000 Nov;14(7):468-73
11113926
Glia. 2000 Sep;31(3):241-8
10941150