Gediminas Vidugiris, Mary Sobol, John Shultz, Jolanta Vidugiriene and Mary J. Cornett1
Promega Corporation, 2800 Woods Hollow Rd., Madison, WI 53711
1IDEX Health & Science LLC, 600 Park Court Rohnert Park, CA 949282
Glutathione (GSH) is involved in cellular protection in several ways and is considered the first line of defense against oxidative damage or stress. Elimination of glutathione can lead to cell death. One measure often used to demonstrate the presence of reactive oxygen species (ROS) in the cell is a change in the ratio of reduced to oxidized glutathione [GSH/GSSG] ratio.
However measuring GSH and GSSG is not without its difficulties. Many current methods require large cell numbers, use reagents that require many steps and protocols that are not easily compatible with automation, such as acid precipitation of cellular proteins, scraping of the cells from growth plates and titration of the solutions to adjust pH.
This poster describes a new method for determining the formation of ROS that can be performed as an “add only” assay. One further advantage of this method is that before GSSG measurement all reduced glutathione is eliminated. This prevents changes in the level of GSSG from the oxidation of GSH in the sample by atmospheric oxygen.
The high sensitivity and specificity of the assay allows the reactions to be performed in a 384 well format very easily. The assay is based on measuring GSH/GSSG levels in the cells using a highly sensitive and accurate luminescence approach.
Here we show how combining the IDEX H&S Innovadyne Nanodrop II dispenser that provides high precision liquid handling with robust luminescence detection system, small changes in cellular ROS response can be detected in less than 200 to 300 hundred cells with high Z’ values.