A fluorescence microplate screen assay for the detection of neurite outgrowth and neurotoxicity using antibodies against βIII‐tubulin

Abstract

The majority of environmental and commercial chemicals have not been evaluated for their potential to cause neurotoxicity. Retinoic acid‐induced neurons from mouse embryonal carcinoma cells is a rapid and sensitive model to assess chemical‐induced neurotoxicity, and we have investigated if neuron specific anti‐βIII‐tubulin antibodies are useful in a microplate assay of neurite outgrowth. By incubating the P19‐derived neurons with the primary anti‐βIII‐tubulin antibody and a secondary Alexa Fluor 488‐conjugated 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 were superimposable to 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 methyl mercury chloride (0.05–1 μM) and okadaic acid (1–50 nM), was significantly higher than a cell viability assay measuring intracellular fluorescence of calcein‐AM. 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 neurotoxicity.