We are working with Caco-2 cells in culture and we need a protocol to measure total glutathione and also superoxide anion production.
Can someone give me the protocol?
Thank you very much!
Dear Romina
There are several kits to measure GSH in cells. The ratio between GSSG and GSH in cells is normaly 1:10 or even lower. GSH makes up 80% of free thiols in cells. Thus determination of free thiols can be used to measure GSH content (however it is not so exact). A kit that works fine is the Fluorometric Thiol Assay Kit from Sigma.
If you want to be more exact determin the ratio between GSH and GSSG. We use the Promega Kit GSH/GSSG-Glo™ Assay. The Merck Millipore Assay: GSH/GSSG Ratio Assay Kit works as well.
Dyes for FACS to determi9ne GSH did not work well in my hands.
Superoxide anions:
In an aqueous solution (cytosol) superoxide anions convert very fast into hydrogen peroxide (half life 1µs, Reth 2002). SODs speed up the reaction. Therefore, it is much easyer to detect hydrogen peroxide (or reactive oxygen speries in total). We use DCFDA and FACS. It should detect hydrogen peroxide but I think it detecs ROS in general.
MitoSox would be an dye that detects superoxide anions in mitochondria. The loading of mitochondria with the dye is membrane potential dependent. Exclude changes in membrane potential by your treatment (e.g. induction of apoptosis).
Best
Karsten
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