Simplified serum- and steroid-free culture conditions for high-throughput viability analysis of primary cultures of cerebellar granule neurons

A serum- and steroid-free primary culture system was developed for the main tenance and automated analysis of cerebellar granule cell viability. Conven tional poly-lysine coated 96-well tissue culture plates serve as a platform for growth, experimental manipulation and subsequent automated analysis of these primary cultured neurons. Cerebellar granule neurons were seeded at densities ranging from 2 x 10(4) to 1.25 x 10(6) cells/cm(2) and maintained in serum- and steroid-free culture conditions for 7 days. Viability was su bsequently determined by the reduction of [3-(4,5-dimethylthiazol-2-yl)-5-( 3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), and the deg ree of cell death occurring over that period was determined by the release of lactate dehydrogenase (LDH). At appropriate cell densities, the results of the MTS reduction and LDH release assays were directly proportional to t he initial number of cerebellar granule cells plated. Those results indicat e that an initial cell density of 0.5-1.0 x 10(5) cells per well (0.32 cm(2 )) was appropriate for simultaneous analysis with the MTS reduction and LDH release assays. Both assays were then used to demonstrate the utility of t his model system for analysis of tert-butyl-hydroperoxide and hydrogen pero xide induced oxidative stress. Additionally, the NITS reduction assay was u sed to demonstrate that the NMDA-receptor selective antagonist MK-801 was n europrotective against glutamate-mediated excitotoxicity. This study define s a powerful and flexible primary culture system for cerebellar neurons tha t is useful for high-throughput analysis of factors that influence neuronal viability. (C) 2001 Elsevier Science B.V. All rights reserved.