A RAPID FLUOROMETRIC ASSAY TO MEASURE NEURONAL SURVIVAL IN-VITRO

We report the development and characterization of a rapid fluorometric microassay suitable for quantifying neuronal cell survival. The method can be used in two formats: (1) a time course analysis of survival response or (2) as a simple endpoint assay for the assessment of neuronal survival promoted by a variety of reagents. The assay uses calcein AM, a non-fluorescent, electrically neutral, non-polar analogue of fluorescein diacetate, which passively crosses cell membranes and is cleaved to a fluorescent derivative by non-specific intracellular esterases. Once cleaved in viable cells, the resultant fluorescent salts are retained by intact cell membranes. The relative number of viable cells under various conditions can be quantified by measuring the emitted fluorescence. Described herein are the conditions that allow the determination of low viable neuronal cell numbers (10(2)-10(3) cells/cm(2)).