琼脂糖明胶皮下埋植法富集、分离具有自发NETs倾向的小鼠中性粒细胞

Recruitment and isolation of NETs-prone mouse neutrophil by subcutaneously implanted agarose gel

目的建立小鼠中性粒细胞富集、分离的方法,为进一步探讨中性粒细胞及中性粒细胞胞外捕网（NETs）的免疫损伤效应奠定基础。方法制备2%琼脂糖0.2%明胶块,埋植于健康6-8周龄C57雌性小鼠皮下富集中性粒细胞,96 h后取出明胶块置于PBS中进行游离释放,收集细胞。荧光染色进行细胞形态学观察,采用Gr-1及CD11b进行流式细胞分析鉴定。结果不同表面积的明胶能够分离出106-107数量级细胞,具有中性粒细胞典型的分叶核形态学特征,流式细胞分析鉴定为Gr-1＋CD11b＋的中性粒细胞,纯度可达95.7%,并观察到典型NETs形成,其数量随分离时间的延长而增加。结论通过皮下埋植琼脂糖明胶成功富集、分离大量高纯度且具有自发NETs倾向小鼠中性粒细胞。

In this study, an improved method of neutrophil recruitment and isolation was established to further study the immunolesion effect of neutrophil and neutrophil extracellular traps（NETs）. 6-8 weeks female C57 mice were subcutaneously implanted with 2% agarose and 0.2% gel blocks for 96 h. Then, the blocks were taken out and placed into PBS for harvesting neutrophils. Cell morphology was observed by fluorescence microscope, and the purity of cells was analyzed by flow cytometry. Data showed that 106-107 neutrophils could be isolated from different area of agarose gel blocks. The purity of Gr-1＋CD11b＋neutrophils was 95.7%, and the cells exhibited classic polymorphonuclear morphology. NETs formation was observed in parts of neutrophils, and the number of NETs was increasing with isolation time went on. In conclusion, agarose gel of subcutaneous implantation in mouse could elicit a large number of high purity NETs-prone neutrophils.