An imaging assay to analyze primary neurons for cellular neurotoxicity.
The development of high-content screening technologies including automated immunostaining, automated image acquisition and automated image analysis have enabled higher throughput of cellular imaging-based assays. Here we used high-content imaging to thoroughly characterize the cultures of primary rat cerebellar granule neurons (CGNs). We describe procedures to isolate and cultivate the CGNs in 96-well and 384-well format, as well as a procedure to freeze and thaw the CGNs. These methods allow the use of CGNs in 96-well format analyzing 2500 samples per experiment using freshly isolated cells. Down-scaling to 384-well format and freezing and thawing of the CGNs allow even higher throughput. A cellular assay with rat CGN cultures was established to study the neurotoxicity of compounds in order to filter out toxic compounds at an early phase of drug development. The imaging-based toxicity assay was able to reveal adverse effects of compounds on primary neurons which were not detected in neuroblastoma or other cell lines tested.
Götte
Marjo
M
Novartis Institutes for BioMedical Research, Forum 1, Novartis Campus, CH-4002 Basle, Switzerland. marjo.goette@novartis.com
Hofmann
Gabriele
G
Michou-Gallani
Anne-Isabelle
AI
Glickman
J Fraser
JF
Wishart
William
W
Gabriel
Daniela
D
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D000941:Antigens / Q000378:metabolism; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism; D002038:Bungarotoxins / Q000633:toxicity; D002452:Cell Count / Q000379:methods; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020128:Inhibitory Concentration 50; D018613:Microscopy, Confocal / Q000379:methods*; D009415:Nerve Net / Q000187:drug effects; D016501:Neurites / Q000187:drug effects; D009447:Neuroblastoma / Q000473:pathology; D009474:Neurons / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D009498:Neurotoxins / Q000494:pharmacology*; D019081:O Antigens / Q000378:metabolism; D009836:Oligodendroglia / Q000187:drug effects / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism; D051381:Rats; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism; D000099216:Chondroitin Sulfate Proteoglycan 4
D000255:Adenosine Triphosphate; D000941:Antigens; D002038:Bungarotoxins; D004121:Dimethyl Sulfoxide; D009498:Neurotoxins; D019081:O Antigens; D011509:Proteoglycans; D014404:Tubulin; D000099216:Chondroitin Sulfate Proteoglycan 4