title: Plate reader-based assays for measuring cell viability, neuroprotection and calcium in primary neuronal cultures.
abstract: Drug discovery and development efforts critically rely on cell-based assays for high-throughput screening. These assay systems mostly utilize immortalized cell lines, such as human embryonic kidney cells, and can provide information on cytotoxicity and cell viability, permeability and uptake of compounds as well as receptor pharmacology. While this approach has proven extremely useful for single-target pharmacology, there is an urgent need for neuropharmacological studies to screen novel drug candidates in a cellular environment resembles neurons in vivo more closely, in order to gain insight into the involvement of multiple signaling pathways. Primary cultured neuronal cells, such as cortical neurons, have long been used for basic research and low-throughput screening and assay development, and may thus be suitable candidates for the development of neuropharmacological high-throughput screening approaches. We here developed and optimized protocols for the use of primary cortical neuronal cells in high-throughput assays for neuropharmacology and neuroprotection, including calcium mobilization, cytotoxicity and viability as well as ion channel pharmacology. Our data show low inter-experimental variability and similar reproducibility as conventional cell line assays. We conclude that primary neuronal cultures provide a viable alternative to cell lines in high-throughput assay systems by providing a cellular environment more closely resembling physiological conditions in the central nervous system.
authors.affiliation: Vision Research Center and Departments of Ophthalmology and Basic Medical Science, University of Missouri - Kansas City, School of Medicine, 2411 Holmes St., Kansas City, MO 64108, United States.
authors.forename: Stephanie L
authors.initials: SL
authors.lastname: Burroughs
authors.forename: R Scott
authors.initials: RS
authors.lastname: Duncan
authors.forename: Parvathi
authors.initials: P
authors.lastname: Rayudu
authors.forename: Prasanthi
authors.initials: P
authors.lastname: Kandula
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Payne
authors.forename: Julie L
authors.initials: JL
authors.lastname: Clark
authors.forename: Peter
authors.initials: P
authors.lastname: Koulen
authors.forename: Simon
authors.initials: S
authors.lastname: Kaja
mesh_terms: D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D057166:High-Throughput Screening Assays / Q000379:methods; D009474:Neurons / Q000166:cytology* / Q000502:physiology*; D009480:Neuropharmacology / Q000379:methods*; D018696:Neuroprotective Agents; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results
chemical_list: D018696:Neuroprotective Agents; D002118:Calcium