title: A fluorescence microplate screen assay for the detection of neurite outgrowth and neurotoxicity using an antibody against βIII-tubulin.
abstract: The majority of environmental and commercial chemicals have not been evaluated for their potential to cause neurotoxicity. We have investigated if neuron specific anti-βIII-tubulin antibodies are useful in a microplate assay of neurite outgrowth of retinoic acid-induced neurons from mouse P19 embryonal carcinoma cells. By incubating the P19-derived neurons with the primary anti-βIII-tubulin antibody and a secondary Alexa Fluor 488-conjugated antibody, followed by measuring the fluorescence in a microplate reader, a time-dependent increase in anti-βIII-tubulin immunofluorescence was observed. The relative fluorescence units increased by 4.3-fold from 2 to 10 days in culture. The results corresponded well with those obtained by semi-automatic tracing of neurites in fluorescence microscopy images of βIII-tubulin-labeled neurons. The sensitivity of the neurite outgrowth assay using a microplate reader to detect neurotoxicity produced by nocodazole, methyl mercury chloride and okadaic acid was significantly higher than for a cell viability assay measuring intracellular fluorescence of calcein-AM. The microplate-based method to measure toxicity targeting neurites using anti-βIII-tubulin antibodies is however less sensitive than the extracellular lactate dehydrogenase activity assay to detect general cytotoxicity produced by high concentrations of clomipramine, or glutamate-induced excitotoxicity. In conclusion, the fluorescence microplate assay for the detection of neurite outgrowth by measuring changes in βIII-tubulin immunoreactivity is a rapid and sensitive method to assess chemical- or toxin-induced neurite toxicity.
authors.affiliation: Department of Pharmacology and Clinical Neuroscience, Umeå University, SE-901 87 Umeå, Sweden.
authors.forename: Dina
authors.initials: D
authors.lastname: Popova
authors.affiliation: Department of Pharmacology and Clinical Neuroscience, Umeå University, SE-901 87 Umeå, Sweden. Electronic address: stig.jacobsson@pharm.umu.se.
authors.forename: Stig O P
authors.initials: SO
authors.lastname: Jacobsson
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D054278:Embryonal Carcinoma Stem Cells / Q000378:metabolism; D005452:Fluoresceins / Q000737:chemistry; D005455:Fluorescent Antibody Technique / Q000379:methods; D008767:Methylmercury Compounds / Q000633:toxicity; D051379:Mice; D008856:Microscopy, Fluorescence; D016501:Neurites / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000473:pathology; D020258:Neurotoxicity Syndromes / Q000175:diagnosis* / Q000209:etiology / Q000473:pathology; D015739:Nocodazole / Q000633:toxicity; D019319:Okadaic Acid / Q000633:toxicity; D013997:Time Factors; D014212:Tretinoin / Q000494:pharmacology; D014404:Tubulin / Q000276:immunology*
chemical_list: D000906:Antibodies; D005452:Fluoresceins; D008767:Methylmercury Compounds; D014404:Tubulin; C462941:beta3 tubulin, mouse; C085925:calcein AM; D019319:Okadaic Acid; D014212:Tretinoin; C004925:methylmercuric chloride; D015739:Nocodazole
keywords: Calcein-AM; Cell culture; Fluorescence; Microplate assay; Neurotoxicity; βIII-tubulin