流式细胞术分选小鼠肺泡巨噬细胞及鉴定

Isolation of mouse alveolar macrophages by flow cytometry and identification

目的建立一种基于流式细胞术分离小鼠肺泡巨噬细胞(AM)的方法。方法小鼠肺脏在体外经胶原酶IV消化后获得单细胞悬液,再经CD11b和CD11c抗体标记,利用流式细胞仪分选获得CD11blowCD11c+AM,并经台盼蓝染色计数细胞活力,以及通过Real-time PCR和吞噬功能进行鉴定。结果流式细胞术分选小鼠肺脏单细胞悬液,获得CD11blowCD11c+细胞,纯度为(93±2)%,细胞活力为(80±5)%;Real-time PCR结果显示过氧化物酶体增殖物激活受体γ(PPARγ)在该群细胞高表达(P<0.001);细胞吞噬功能实验结果显示该群细胞具有较好的吞噬活性,吞噬率约为10%。结论建立了一种基于流式细胞术分选获得AM的方法,该方法获得的细胞纯度高,细胞活性好,可用于功能性实验。

Objective To establish a method for isolating alveolar macrophage （AM） of mouse based on flow cy- tometry. Methods The lungs were digested by collagen IV in vitro to prepare single-cell suspension that was stained by CD1 l b and CD11 c antibody. CD11 bIwCD11 c cell population were AM and isolated by flow cytometry. After that, the cell viability was measured via the Typan blue staining, and the identification of AM was through flow cytometry and real-time PCR. Results CD1 l bWCD1 l c cell population was isolated by flow cytometry, the purity was （93 ± 2）% and the cell viability was （80 ＋ 5 ）%. The real-time PCR results showed that peroxisome proliferator-activated receptor γ（PPARγ） mRNA was highly expressed in AM isolated by flow cytometry （P 〈 0. 001 ）. In addition, the functional assay showed that the isolated AM possess high phagocytic activity. Thus, the results described above demonstrate that the isolated cells were AM. Conclusion A method for obtaining AM based on flow cytometry was established. The method has high cell purity and good cell activity which can be used for functional experiments.