OPTIMIZATION OF TWO METHODS FOR ASSESSING CELL VIABILITY AND CYTOPROTECTION IN C6 ASTROCYTES

Purpose: The purpose of this study was to determine the best method for measuring cell viability in the rat C6 astrocyte cell model in response to two cytotoxic insults, hydrogen peroxide (H2O2) and iodoacetic acid (IAA). Methods: Two assays were evaluated: calcein-AM assay for detecting live cell number, and a flow cytometry-based assay to assess live versus dead populations. Cells were treated with 0, 10, 20 or 50 μM H2O2 (2 hours) or IAA (3 hours). The calcein-AM assay was evaluated in a 96-well plate format. Flow cytometry results were obtained using a C6 Accuri model Flow Cytometer, and cells were stained with both calcein-AM (peak emission range 485-535 nm, fluorescence channel 1 (FL1)) and ethidium homodimer (peak emission range 610-30 nm, fluorescence channel 3 (FL3)). Results: Treated cells displayed a concentration dependent decrease in cell viability (calculated EC50 = 18μM for H2O2, 23 μM for IAA). Cells treated for 3 hours with IAA showed a concentration dependent transition from high FL1, low FL3 fluorescence to low FL1, high FL3 fluorescence, indicating a transition from high to low viability. Conclusions: This study describes two methods for cell viability detection: calcein fluorescence by high-throughput analysis and simultaneous calcein and ethidium homodimer staining for flow cytometry cell gating for individual cell analysis. Having successfully utilized both the 96-well assay and the flow cytometry protocols to assess cell viability, we plan to extend the current studies by assessing how brain-active steroids protect glia from insults relevant to brain aging and certain neurodegenerative diseases.