一种优化小鼠成纤维细胞中自噬小体示踪的方法

Optimization of a method to trace the autophagosomes in mouse fibroblasts

目的为了提高对自噬小体示踪的精确度和清晰度,全面展示细胞自噬相关进程,优化基于绿色荧光蛋白-微管相关蛋白1轻链3β(GFP-LC3β)标记物的自噬小体示踪方法。方法组织块贴壁法原代培养小鼠耳成纤维细胞,脂质体转染过表达GFP-LC3β;GFP-LC3β充分表达后给予药物刺激以激活或阻断细胞自噬通路,然后进行免疫荧光染色。对染色操作进行优化:首先,延长多聚甲醛的固定细胞时间(从15min延长至45min);其次,提高Triton X-100处理时间,除第一次和最后一次使用PBS润洗,其余每一次使用的PBS都添加0.2%Triton X-100;最后,提升抗体稀释液中BSA和Triton X-100含量,BSA从3%提升至5%,TritonX-100的含量从0.1%提升至0.2%。结果拍照后统计自噬小体发现,新方法更加准确地标记GFP-LC3β指示物,自噬激活条件下示踪准确度显著提升(达4.2倍);新方法更加清晰地呈现自噬小体的形态和胞内分布,将自噬小体示踪的清晰度显著提升(达2.5倍);新方法还能使用不同荧光通道,同时标记其他自噬相关基因。结论本研究提供的一种优化小鼠成纤维细胞中自噬小体示踪的方法将为全面展示细胞自噬进程、深入了解细胞自噬相关机制提供有力的技术支持。

Objective To improve the accuracy and clarity of autophagosome tracing, fully reveal the process of autophagy, and optimize the autophagosome tracing method based on （ green fluorescent protein-light chain 3β of micmtubule associate protein 1, GFP- LC3β）. Methods Mouse ear fibroblasts were obtained from primary adherent culture of tissue explants. GFP-LC3β was transfected by lipofection and over-expressed. After GFP-LC3β was fully expressed, rapamycin or chloroquine was applied to stimulate or block the autophagy pathway. Immunofluorescent staining was performed afterwards. The staining strategy was optimized as below： （1） the time of paraformaldehyde （PFA） treatment was extended from 15min to 45 min; （2） 0. 2% Triton X-100 was added to PBS except in the first and last rinses; （3） the concentrations of BSA and Triton X-100 in the antibody diluent were increased from 3% to 5% and from 0. 1% to 0. 2%, respectively. Results Autophagosomes were counted and analyzed after image capturing, from which it was found that the optimized procedure labeled GFP-LC313 more accurately and displayed the morphology and distribution of autophagosomes more clearly; the accuracy of tracing was increased by 4. 2 times when autophagy was activated while the clarity was increased by 2. 5 times. In addition, more alternative fluorescent channels were accessible to other aut@agic genes with the new method. Conclusion This op- timized procedure for tracing autophagosomes in mouse fibroblasts facilitates a comprehensive display of the autophagic process and pro- vides expertise technical support for a better understanding of autophagic mechanisms.