小鼠免疫细胞流式染色过程中细胞丢失量的影响因素
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摘要
目的观察和探讨小鼠免疫细胞多色流式染色过程中细胞丢失的影响因素。方法取C57BL/6J鼠制备脾细胞悬液,分别用不同种类、不同成分和不同pH值缓冲液洗涤和重悬细胞2次,并计数活细胞数量,观察对染色过程中的细胞丢失量有明显影响的因素。结果在小鼠免疫细胞的流式染色标记过程中,缓冲液种类对细胞丢失量有显著影响,含有钾离子的PBS缓冲液洗涤后会造成明显的细胞丢失;离心机转速过高或过低均会导致显著的细胞丢失;而缓冲液pH值的微量偏移和是否加入FBS/BSA对细胞的丢失影响不明显。结论小鼠免疫细胞的流式染色过程中使用含有钾离子的PBS缓冲液会造成明显的细胞丢失,应推荐使用不含钾离子的PBS缓冲液,同时保持离心转速适中。
Objective To explore the factors affecting cell loss in multicolor flow cytometry of mouse immune cells.Methods C57BL/6Jmice were used in the experiment.Splenocytes were isolated and resuspended twice with buffers of different types,compositions and pH;then the number of viable cells was counted to observe which factors affected the amount of cell loss during staining.Results During flow cytometric labeling of mouse immune cells,buffer types significantly affected the amount of cell loss.Washing with PBS buffer containing potassium ions caused obvious cell loss,while slight shift in buffer pH and whether or not FBS/BSA was added had no significant effect on cell loss.Moreover,centrifugation speed also significantly affected cell loss.Conclusion The use of potassiumcontaining PBS buffer during flow cytometric staining of mouse immune cells resulted in significant cell loss.The potassium-free PBS buffer and moderate centrifugation speed should be recommended.
Objective To explore the factors affecting cell loss in multicolor flow cytometry of mouse immune cells.Methods C57BL/6Jmice were used in the experiment.Splenocytes were isolated and resuspended twice with buffers of different types,compositions and pH;then the number of viable cells was counted to observe which factors affected the amount of cell loss during staining.Results During flow cytometric labeling of mouse immune cells,buffer types significantly affected the amount of cell loss.Washing with PBS buffer containing potassium ions caused obvious cell loss,while slight shift in buffer pH and whether or not FBS/BSA was added had no significant effect on cell loss.Moreover,centrifugation speed also significantly affected cell loss.Conclusion The use of potassiumcontaining PBS buffer during flow cytometric staining of mouse immune cells resulted in significant cell loss.The potassium-free PBS buffer and moderate centrifugation speed should be recommended.
引文
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[2]MCNELIS JC,OLEFSKY JM.Macrophages,immunity,and metabolic disease[J].Immunity,2014,41(1):36-48.
[3]COSMI L,MAGGI L,SANTARLASCI V,et al.T helper cells plasticity in inflammation[J].Cytometry A,2014,85(1):36-42.
[4]HIRAHARA K,NAKAYAMA T.CD4+T-cell subsets in inflammatory diseases:Beyond the Th1/Th2paradigm[J].Int Immunol,2016,28(4):163-171.
[5]POCKLEY AG,FOULDS GA,OUGHTON JA,et al.Immune cell phenotyping using flow cytometry[J].Curr Protoc Toxicol,2015,66:18.8.1-34.
[6]MACIOROWSKI Z,CHATTOPADHYAY PK,JAIN P.Basic multicolor flow cytometry[J].Curr Protoc Immunol,2017,117:5.4.1-5.4.38.
[7]WIEDE F,TIGANIS T.Isolation and characterization of mouse intrahepatic lymphocytes by flow cytometry[J].Methods Mol Biol,2018,1725:301-311.
[8]JAIMES MC,MAECKER HT,YAN M,et al.Quality assurance of intracellular cytokine staining assays:Analysis of multiple rounds of proficiency testing[J].J Immunol Methods,2011,363(2):143-157.