| 何静怡，舒鼎铭，林楚晓，李 莹，王 艳，罗成龙.鸡树突状细胞的间接免疫荧光制片方法比较[J].广东农业科学,2020,47(10):126-131 |
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鸡树突状细胞的间接免疫荧光制片方法比较 |
| Comparison of Slide-preparing Methods of Chicken Bone Marrow Dendritic Cells by Indirect Immunofluorescence |
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| DOI：10.16768/j.issn.1004-874X.2020.10.016 |
| 中文关键词: 鸡 树突状细胞 免疫荧光 激光共聚焦扫描 细胞爬片法 细胞滴片法 |
| 英文关键词: chicken dendritic cell immunofluorescence laser scanning confocal microscopy cell climbing cell dropping |
| 基金项目:国家自然科学基金（31402067）；广东省自然科学基金（2018B030311044）；国家现代农业（肉鸡）产业技术体系专项（CARS-41）；“广东特支计划”科技创新青年拔尖人才项目（2015TQ01N843）；广东省农业科学院科技创新战略专项资金（高水平农科院建设）- 人才项目（R2017PY-QY007） |
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| 中文摘要: |
| 【目的】比较分析骨髓源鸡树突状细胞两种间接免疫荧光制片方法（细胞爬片法和细胞滴片法），以进一步提高研究树突状细胞生物学的实验技术和方法。【方法】以鸡胚为试验素材，分离原代细胞，定向诱导分化获得未成熟的树突状细胞，并利用新城疫病毒（newcastle disease virus，NDV）lasota 株激活细胞。监测内源性蛋白 ROBO2（Roundabout）和外源性蛋白 NDV 表达情况，鸡源 NDV 抗体与小鼠源 ROBO2 单克隆抗体混合孵育，结合荧光素二抗进行标记。应用激光共聚焦扫描显微镜（laser scanning confocal microscopy, LSCM）对树突状细胞进行荧光成像并捕抓图像数据，多方位分析对比细胞爬片法和细胞滴片法。【结果】细胞滴片制作过程繁琐且耗时长；细胞爬片则操作简单可缩短实验时间。两种制片方法的 NDV 和 ROBO2 单阳性和阴性分别存在极显著差异，双阳性存在荧光反应性显著差异。【结论】滴片法蛋白免疫反应性更优，适合标记目的蛋白进行亚细胞定位。爬片法始终维持细胞形态学特征和保持蛋白特性，利于两抗体荧光重叠，综合显色性更强。 |
| 英文摘要: |
| 【Objective】In order to improve the laboratory techniques and methods of dendritic cells（DC）, two slide-preparing methods（cell climbing and cell dropping）of DC from chicken bone marrow by indirect immunofluorescence was compared and analyzed.【Method】Using chicken embryos as test material, the immature bone marrow cells were isolated and obtained from chicken in vitro, and then were activated by Newcastle disease virus（NDV） lastota strain. The expressions of endogenous protein ROBO2（Roundabout） and exogenous protein NDV were monitored. Chicken NDV antibody and mouse ROBO2 monoclonal antibody were hatched together and marked with the combination of FITC. Fluorescence imaging of dendritic cells was examined by laser scanning confocal microscopy（LSCM）, and the difference between cell climbing and cell dropping was analyzed variously.【Result】The cell dropping process was time- consuming and labor intensive, while the cell climbing process was characterized by easy operation and reduced time. NDV and ROBO2 of these two methods showed highly significant differences in single positive and negative and significant influorescence reactivity differences in double positive.【Conclusion】The immunoreactivity of the protein was better with the cell dropping, which was suitable for labeling target and subcellular localization. The morphological and protein characteristics were always
maintained by the cell climbing,which was beneficial to the fluorescence overlap of the two antibodies and to make the overall color rendering stronger. |
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