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 metho d can be used in two formats: (1) a time course analysis of survival r esponse or (2) as a simple endpoint assay for the assessment of neuron al survival promoted by a variety of reagents. The assay uses calcein AM, a non-fluorescent, electrically neutral, non-polar analogue of flu orescein diacetate, which passively crosses cell membranes and is clea ved to a fluorescent derivative by non-specific intracellular esterase s. Once cleaved in viable cells, the resultant fluorescent salts are r etained by intact cell membranes. The relative number of viable cells under various conditions can be quantified by measuring the emitted fl uorescence. Described herein are the conditions that allow the determi nation of low viable neuronal cell numbers (10(2)-10(3) cells/cm(2)).