An optimized method for determination of intracellular glutathione in mouse macrophage cultures by fluorimetric high-performance liquid chromatography.
We have optimized a method for the determination of intracellular glutathione by high-performance liquid chromatography, using fluorimetric detection. To minimize artifacts and provide an accurate determination of intracellular glutathione, cell extracts were prepared using extraction conditions specifically designed to inhibit autoxidation and enzymatic degradation of glutathione. The sensitivity of the method was enhanced by adjusting the dansyl chloride derivatization reaction with regard to parameters such as pH, reaction time and dansyl chloride concentration. Both oxidized and reduced forms of glutathione were quantified using the refined method in extracts of oxidatively stressed J774A.1 mouse macrophage cells and reflected an expected shift in cellular redox status.
Komatsu
Hiroyoshi
H
Department of Immunology, School of Allied Health Sciences, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan. hkomatsu@cc.ahs.kitasato-u.ac.jp
Obata
Fumiya
F
D000818:Animals; D002457:Cell Extracts; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003619:Dansyl Compounds / Q000737:chemistry; D005978:Glutathione / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D016676:Macrophages, Alveolar / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D010084:Oxidation-Reduction; D010472:Perchlorates / Q000494:pharmacology; D012015:Reference Standards; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D002457:Cell Extracts; D003619:Dansyl Compounds; D010472:Perchlorates; D005978:Glutathione