Premium
A fluorescence microplate screen assay for the detection of neurite outgrowth and neurotoxicity using antibodies against βIII‐tubulin
Author(s) -
Popova Dina,
Jacobsson Stig O.P.
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.1046.2
Subject(s) - neurite , alexa fluor , neurotoxicity , tubulin , chemistry , microbiology and biotechnology , fluorescence , immunofluorescence , antibody , biochemistry , biology , microtubule , in vitro , toxicity , immunology , physics , organic chemistry , quantum mechanics
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.