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Quantification of green fluorescent protein in cellular supernatant by capillary electrophoresis with laser-induced fluorescence detection for measurement of cell death
Authors:Kristian E. Swearingen  Benjamin Kehimkar  Norman J. Dovichi
Affiliation:a Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195-1700, United States
b Departments of Laboratory Medicine and Microbiology, University of Washington, Box 357110, Seattle, WA 98195-7110, United States
Abstract:A common method for quantifying cell death is measuring the concentration of lactate dehydrogenase (LDH) released by cells as their membranes become unstable. In cells expressing green fluorescent protein (GFP), degradation of the cell membrane also results in the release of GFP into the surrounding supernatant. In this study, we used capillary electrophoresis with laser-induced fluorescence detection to measure the levels of GFP in supernatants of UBIGFP/BL6 primary macrophages that had been infected with Salmonella typhimurium, treated with staurosporine, or exposed to H2O2, all known inducers of cell death. We also used a standard LDH assay to measure the release of LDH into supernatants. We observed the rate of cell death quantified by release of GFP and LDH into supernatant to be essentially identical, demonstrating that GFP release is at least as good as an indicator of macrophage cell death as the established LDH release method.
Keywords:Capillary electrophoresis   Laser-induced fluorescence   Green fluorescent protein   Cell death   Lactate dehydrogenase assay
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