A kinetic microassay for glutathione in cells plated on 96-well microtiter plates.
We describe a modified enzymatic, kinetic, glutathione microassay based on the original macroassay described by Tietze and modified by Anderson. It is coupled with the Triton X-100 lysis and an acid extraction that can be performed in 96-well microtiter plates. The microassay can be read in a microplate reader equipped with a standard 405 nm filter. Intracellular glutathione levels are not significantly different when comparing the proposed Triton X-100 lysis and acid extraction method from those found with the cellular homogenization and acid extraction method typically employed. By combining a rapid sample extraction method, which can be done on the microtiter plate, with an assay based on the technology of a microplate reader, we have devised a rapid, reproducible, inexpensive and easy to use GSH microassay that can process several hundred samples daily and will be useful in studies where many replicate samples are required. We have successfully used this method to monitor glutathione status in a human cell line exposed to organochlorine pesticides and cells exposed to naturally occurring mixtures of metals extracted from aquatic sediments.
Allen
S
S
Biology, Canisius College, Buffao, New York 14208, USA.
Shea
J M
JM
Felmet
T
T
Gadra
J
J
Dehn
P F
PF
D006528:Carcinoma, Hepatocellular / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D005978:Glutathione / Q000032:analysis* / Q000187:drug effects; D006801:Humans; D007306:Insecticides / Q000633:toxicity; D008113:Liver Neoplasms / Q000737:chemistry; D015203:Reproducibility of Results; D013439:Sulfhydryl Reagents / Q000737:chemistry; D014407:Tumor Cells, Cultured / Q000378:metabolism
D007306:Insecticides; D013439:Sulfhydryl Reagents; D004228:Dithionitrobenzoic Acid; D005978:Glutathione