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Fluorometric Estimation of Glutathione in Cultured Microglial Cell Lysate
Vikas Singh1,2, Ruchi Gera1,2, Mahaveer Prasad Purohit3,2
1Immunotoxicology Laboratory, Food Drug and Chemical Toxicology Group and Nanotherapeutics & Nanomaterial Toxicology Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), 31-Vishvigyan Bhawan, MG Marg, Lucknow, Uttar Pradesh-226001, India.
Abstract:
Glutathione is one of the major antioxidant defense components present in cells. It is predominantly present as reduced glutathione (GSH) and converted into oxidized glutathione (GSSG) while reducing the free radicals like hydroxyl ions (OH-). For the measurement of GSH and GSSG, o-phthalaldehyde (OPT) has been used as a fluorescent reagent. O-phthalaldehyde has an ability to react specifically with GSH at pH 8 and GSSG at pH 12 respectively. N-ethylmaleimide (NEM) has been used to prevent auto-oxidation of GSH during measurement of GSSG in the present protocol. The original protocol by Hissin and Hilf was developed for glutathione estimation in Rat liver tissue. The present protocol has been standardized following Hissin and Hilf (1976) for the estimation of glutathione in cultured microglial cell lysate but it can also be used for other mammalian cell lysate. In our lab same protocol has been used for the estimation of glutathione in the whole cell lysate of murine neuroblastoma cell, N2a.
