Popova Dina, Jacobsson Stig O P
Department of Pharmacology and Clinical Neuroscience, Umeå University, SE-901 87 Umeå, Sweden.
Department of Pharmacology and Clinical Neuroscience, Umeå University, SE-901 87 Umeå, Sweden.
Toxicol In Vitro. 2014 Apr;28(3):411-8. doi: 10.1016/j.tiv.2013.12.009. Epub 2013 Dec 31.
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.
大多数环境化学品和商用化学品尚未就其导致神经毒性的可能性进行评估。我们研究了神经元特异性抗βIII-微管蛋白抗体在检测从小鼠P19胚胎癌细胞诱导分化的神经元轴突生长的微孔板试验中是否有用。通过将源自P19的神经元与抗βIII-微管蛋白一抗和荧光素标记的二抗孵育，然后在微孔板读数仪中测量荧光，观察到抗βIII-微管蛋白免疫荧光随时间增加。培养2至10天，相对荧光单位增加了4.3倍。该结果与通过对βIII-微管蛋白标记的神经元的荧光显微镜图像中的轴突进行半自动追踪所获得的结果非常吻合。使用微孔板读数仪检测诺考达唑、甲基氯化汞和冈田酸产生的神经毒性的轴突生长试验的灵敏度显著高于测量钙黄绿素-AM细胞内荧光的细胞活力试验。然而，使用抗βIII-微管蛋白抗体基于微孔板测量针对轴突的毒性的方法，比细胞外乳酸脱氢酶活性试验检测高浓度氯米帕明产生的一般细胞毒性或谷氨酸诱导的兴奋性毒性的灵敏度要低。总之，通过测量βIII-微管蛋白免疫反应性变化来检测轴突生长的荧光微孔板试验是一种评估化学物质或毒素诱导的轴突毒性的快速且灵敏的方法。
