An automated colorimetric microassay for neuronotrophic factors.

Abstract

A microassay is described for determining the number of neurons surviving after 24 h in response to added neuronotrophic factors. Neuronal cultures in 96-well microtiter plates are supplied with a yellow tetrazolium derivative, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), which is taken up selectively by viable neurons and converted to a blue formazan product. The amount of blue color development can be rapidly quantified using an automatic microplate spectrophotometer. The resulting optical density is directly proportional to the number of viable neurons. The spectrophotometer has been interfaced with a computer allowing a print out of individual absorbance values and calculation of half-maximal (one trophic unit) neuronal survival. The assay has been used for the quantification of the trophic activities of nerve growth factor and ciliary neuronotrophic factor using, respectively, dorsal root and ciliary ganglionic neurons from 8-day chick embryos. Assay parameters were optimized so that about 2000 individual cultures of ganglionic neurons can be set up and analyzed each day, thus allowing the serial titration in duplicate of 80-120 separate samples. The determination of neuronal number and titer calculation steps now requires about 2 min per microplate (96 cultures), a 50-fold reduction in time over existing methods.