title: Simplified serum- and steroid-free culture conditions for high-throughput viability analysis of primary cultures of cerebellar granule neurons.
abstract: A serum- and steroid-free primary culture system was developed for the maintenance and automated analysis of cerebellar granule cell viability. Conventional 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 subsequently determined by the reduction of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), and the degree 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 the initial number of cerebellar granule cells plated. Those results indicate 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 this model system for analysis of tert-butyl-hydroperoxide and hydrogen peroxide induced oxidative stress. Additionally, the MTS reduction assay was used to demonstrate that the NMDA-receptor selective antagonist MK-801 was neuroprotective against glutamate-mediated excitotoxicity. This study defines a powerful and flexible primary culture system for cerebellar neurons that is useful for high-throughput analysis of factors that influence neuronal viability.
authors.affiliation: Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Mail Slot 611, 4301 West Markham Street, Little Rock, AR 72205, USA.
authors.forename: J K
authors.initials: JK
authors.lastname: Wong
authors.forename: P R
authors.initials: PR
authors.lastname: Kennedy
authors.forename: S M
authors.initials: SM
authors.lastname: Belcher
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002470:Cell Survival / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D002525:Cerebellar Cortex / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D004396:Coloring Agents / Q000378:metabolism; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005260:Female; D018698:Glutamic Acid / Q000633:toxicity; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D009482:Neurophysiology / Q000295:instrumentation / Q000379:methods*; D018696:Neuroprotective Agents / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013256:Steroids / Q000494:pharmacology; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism
chemical_list: D004396:Coloring Agents; D016895:Culture Media, Serum-Free; D018691:Excitatory Amino Acid Antagonists; D018696:Neuroprotective Agents; D013256:Steroids; D013778:Tetrazolium Salts; D013844:Thiazoles; D018698:Glutamic Acid; D007770:L-Lactate Dehydrogenase; C022616:thiazolyl blue