Anne T. Ferguson, Ph.D.
Molecular Devices Corporation
This application note describes the use of the SPECTRAmax GEMINI XS from Molecular Devices to study the performance of the LIVE/DEAD Viability/Cytoxicity Assay Kit for animal cells. This assay uses two fluorescent dyes, calcein AM (cal AM) and ethidium homodimer (EthD-1), to stain live and dead cells simultaneously. Cal AM is an electrically neutral, nonfluorescent, esterase substrate that diffuses into live cells and becomes enzymatically cleaved by ubiquitous cytoplasmic esterases. This releases the free calcein fluorophore that is retained inside live cells. The dye emits a strong green fluorescence that peaks at ~525nm when excited at ~485nm. In contrast, EthD-1 is a polar nucleic acid stain that can penetrate dead, but not live cell membranes. Once intercalated into nucleic acids, it produces a 40-fold increase in red fluorescence at ~625 when excited at ~525.
The present study first determines the optimal excitation (Ex) and emission (Em) wavelengths for each dye. Next, these Ex and Em wavelengths are used to determine how well the two dyes are distinguished in pure populations of live and dead cells in order to predict interference when both dyes are present.
LIVE/DEAD Viability/Cytotoxicity is a sensitive assay designed to monitor changes in the ratio of live to dead cells in the total cell population. Consequently, it is useful for screening compounds that induce cell death, as well as a basic research tool for studying apoptosis, cell-mediated immunity and cell proliferation (1-7). Some advantages of LIVE/DEAD Viability/Cytotoxicity are that live cell staining is not dependent on cell metabolism or proliferation as in the MTT and thymidine uptake assays, and it is a nonradioactive assay unlike thymidine uptake and 51Cr release assays (8, 9).