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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。
OBJECTIVE: To establish a method for the enrichment and isolation of neutrophils in mice and lay the foundation for further study on the immune damage effects of neutrophils and neutrophil extracellular network (NETs). Methods 2% agarose 0.2% gelatin block was prepared and implanted into healthy C57 female mice aged 6-8 weeks to enrich for neutrophils subcutaneously. After 96 hours gelatin blocks were removed and placed in PBS for free release and the cells were harvested. Fluorescent staining was used to observe the morphology of the cells, and Gr-1 and CD11b were used to identify the cells by flow cytometry. Results Gelatin with different surface area could separate 106-107 cells with morphological features typical of neutrophils and was identified as Gr-1 + CD11b + neutrophils by flow cytometry with a purity of 95.7% , And the formation of typical NETs was observed, the number of which increased with the separation time. Conclusion A large number of neutrophils with high purity and spontaneous NETs tendency were isolated by subcutaneous implantation of agarose gelatin.