SELECTIVE LABELING OF EMBRYONIC NEURONS CULTURED ON ASTROCYTE MONOLAYERS WITH 5(6)-CARBOXYFLUORESCEIN DIACETATE (CFDA)

A method for selectively labeling cultured neurons using the vital dye, 5(6)-carboxyfluorescein diacetate (CFDA), is described. This non-fluorescent membrane-permeant dye is cleaved by cytosolic esterases into the fluorescent anion, 5(6)-carboxyfluorescein (CF). Both astrocytes and neurons exhibit brilliant fluorochromasia within minutes of CFDA loading. However, following a brief rinse in buffered saline in the absence of CFDA, the astrocytes rapidly lose their cellular fluorescence while the neurons retain the dye for several hours. The fluorochromasia is uniformly distributed throughout the soma and processes which greatly facilitates the morphological identification of viable neurons. In addition, this protocol can be used to conveniently quantify neuronal survival in assays of the activities of neurotrophic or neurotoxic substances.