Comparative Study on Three Methods for Isolating Mononuclear Cells from Mouse Lung Tissue
This study comprehensively compared the effects of three different methods for isolating mononuclear cells from mouse lung tissue,and provided an effective method for isolating the subpopulations of mouse lung immune cells.Fifteen female C57BL/6 mice(6-8 weeks old)were randomly divided into three groups(n=5/group),including the grinding with lymphocyte separation solution gradient centrifugation group(Ml group),collagenase Ⅳ digestion with lymphocyte separation solution gradient centrifugation group(M2 group),and collagenase Ⅳ digestion group(M3 group).The mice were sacrificed by cervical dislocation,and their lungs were extracted.The suspension of lung mononuclear cells was prepared by the above three methods,and photographed and counted under the microscope.The proportion and number of different immune cell subsets in lung tissues were detected by flow cytometry.Statistical analysis was conducted using SPSS 26.0 software.The results showed that the number of lung mononuclear cells in the M3 group was significantly higher than that in the M1 and M2 groups(F=44.49,P＜0.001).the proportion of NK(F=16.68,P＜0.001),CD4+T(F=12.28,P＜0.01)and B cells(F=4.49,P＜0.05)in lung mononuclear cells in M2 group were higher than that in the M1 and M3 groups,and the proportion of CD3+T,MDSC,neutrophils and lung interstitial macrophages prepared in M3 group was significantly higher than that in the M1 and M2 groups;the number of NK cells,CD3+T,CD4+T,CD8+T,B cells,and myeloid cells(MDSC,neutrophils,DC and macrophages)prepared in M3 group were higher than that in the M1 and M2 groups.M3 group obtained the highest yield of lung mononuclear cells in mouse,and this method could effectively separate the lung NK,T,B lymphocytes and subpopulations of myeloid derived immune cells.This study provides an experimental basis for the immunological pathogenesis of lung re-lated diseases.
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