FACSAriaⅢ流式细胞仪分选小鼠肺脏自然杀伤细胞方法的建立与评价

Establishment and Evaluation of A Method for Sorting Mouse Lung Natural Killer Cells by FACSAriaⅢFlow Cytometer

目的利用流式细胞仪分选小鼠肺脏自然杀伤细胞(NK细胞)并检测分选后细胞纯度与存活率。方法小鼠肺脏经胶原酶灌注法得到单细胞悬液,流式细胞仪分选悬液中FVS510-CD3-CD19-NK1.1+NKp46+细胞,分选后的细胞流式细胞仪回测阳性率,台盼蓝染色检测细胞活力。结果第一滴液流断点位置Drop1为345,Gap为9,液流开启0.5 h以上无晃动,无断点位置改变,稳定性良好,各种参数符合分选条件。分选前NK细胞占总细胞(4.40±1.20)%Total,分选后为(96.10±2.20)%Total,差异有统计学意义(P<0.05)。经台盼蓝染色显示,分选后细胞存活率为(95.70±2.02)%,细胞形态良好。结论利用流式细胞分选仪可快速高效分离小鼠肺脏NK细胞,分选后的细胞保持良好的活性,可为进一步研究NK细胞提供保证。

Objective To sort natural killer cells(NK cells)from mouse lungs by flow cytometry and to detect the purity and survival rate of the cells after sorting.Methods Mouse lungs were perfused with collagenase to obtain a single cell suspension,and FVS510-CD3-CD19-NK1.1+NKp46+cells in the suspension were sorted by flow cytometry.After sorting,flow cytometry back-tested the positive rate of cells,and trypan blue staining to detect cell viability.Results The breakpoint position of the first drop flow was Drop1 was 345,Gap was 9,and the flow was turned on for more than 0.5 h without shaking,no breakpoint position change,good stability,and various parameters were in line with the sorting conditions.Before sorting,NK cells accounted for(4.4±1.2)%Total,After sorting,it was(96.10±2.20)%Total,the difference was statistically significant(P<0.05).The trypan blue staining showed that the cell survival rate after sorting was(95.70±2.02)%,and the cell morphology was good.Conclusion The flow cytometer can quickly and efficiently isolate NK cells from mouse lungs,and the sorted cells maintain good viability,which can provide a guarantee for further research on NK cells.