22.214.171.124. Glutathione estimation
The sulfhydryl group of GSH is reduced by 5-5'-dithiobis (2-nitrobenzoic acid) (DTNB) to form the yellow color product 5-thio-2-nitrobenzoic acid (TNB). The absorbance of TNB is measured at 412 nm (Figure 2.18). Figure 2.18. Schematic of the principle of GSH estimation
Free sulphydryl content was measured as described by Ellman (1959) . Briefly, cells were exposed to different concentrations of ENPs for 6 h. Thereafter, the cells were sonicated in 0.5 ml of 1 X PBS.
To 0.5 ml of homogenate, 0.5 ml of 5% tri-chloro acetic acid (TCA) was added and centrifuged at 845 x g for 15 min at 4◦C. Further, 0.5 ml of the supernatant was added to 2.5 ml of 0.01% DTNB and the absorbance was read at 412 nm (Figure 2.19). The protein present in the pellet was determined by the Bradford method (Bradford, 1976) and the concentration of GSH was expressed as mmol/mg protein. Different concentrations of glutathione were taken as standard and data was represented as μmoles/mg protein.…show more content… Flowchart showing the protocol of GSH estimation
126.96.36.199. Nitric oxide determination
Nitric oxide (NO) was estimated in cell culture medium as stable nitrite using Greiss reagent as described by Green et al (1982). It is based on the azo dye formation from diazonium salt (formed from reaction between sulfanilic acid and nitrite) coupling to naphthylethylenediamine dihydrochloride (Figure 2.20). Figure 2.20. Schematic representation of mechanism of Greiss reagent
Reagent A- 2% Sulphanilamide in 5%