Protocols

Description

Fluorescence based asssay for establishing neuronal viability in cell culture monolayers.

Procedure

Monolayer cultures of rat embryonic cortical neurons were prepared and plated at a density of 2-5 x 105 cells per well in 24 well plates which were precoated with poly-L-lysine (Becton-Dickinson Labware, Bedford, MA) and incubated in the presence or absence of test agents for 2 days at 37C in a 5% CO2 incubator. Immunostaining with an anti-neurofilament polyclonal antibody (N-4142; Sigma, St. Louis, MO) demonstrated that approximately 80-90% of the cells present were neurons.

Neuronal viability was assessed with the fluorescent viability dye, calcein-AM (Molecular Probes, Eugene, OR. Briefly, neuronal cultures were treated with test agents for the indicated times, after which the monolayers were washed once with 1 ml of phosphate-buffered saline (PBS) and then incubated for 20 min at 37C with 1 μM calcein-AM in PBS. The cell monolayers were then washed twice with 1 ml of PBS and the cellular fluorescence remaining on the plate was determined with a fluorescent plate reader (CytoFluor II, Persceptive Biosystems, MA) with excitation at 485 nm and emission at 530 nm. The resulting data were analyzed using GraphPad Prism 3.0 (GraphPad Software, Inc, San Diego, CA). Neuronal cultures examined by fluorescence microscopy confirmed that the calcein AM fluorescence accurately reflected the number of viable neurons present in the culture dish.

Recipes

Calcein AM (Molecular Probes,OR)1 mM in DMSO. Dilute toa final conentration of 1 micromolar in cell culture growth medium.

Supplies

Fluorescence plate reader; standard cell culture facilities, supplis and reagents.

Tips

Perform a timecourse and concetration-response curve to determine the optimized assay conditions.