A fluorescence microplate screen assay for the detection of neurite outgrowth and neurotoxicity using an antibody against βIII-tubulin
2014 (English)In: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 28, no 3, 411-418 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
Elsevier, 2014. Vol. 28, no 3, 411-418 p.
neurotoxicity, cell culture, βIII-tubulin, beta III-tubulin, calcein-AM, fluorescence, microplate assay
Pharmacology and Toxicology
IdentifiersURN: urn:nbn:se:umu:diva-87628DOI: 10.1016/j.tiv.2013.12.009ISI: 000332053800010OAI: oai:DiVA.org:umu-87628DiVA: diva2:711635