应用流式细胞技术分选单核细胞亚群不同预富集方法的探讨
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摘要
目的探讨不同样本预富集方法对流式细胞术分选单核细胞的影响。方法采集15例志愿者外周血,分别采用红细胞裂解法、羟乙基淀粉离心沉淀法和Ficoll密度梯度离心法进行样本预富集处理。台盼蓝染色法检测细胞活力,流式细胞术对单核细胞进行绝对计数并对各亚群比例进行分析。结果 3种方法制备样本的细胞活力均达到95%以上;红细胞裂解法与未进行预富集样本的单核细胞绝对计数和分群无明显差别,羟乙基淀粉法细胞分群不明显,Ficoll密度梯度离心法获取的单核细胞数量明显低于未进行预富集样本。结论外周血单核细胞流式分选技术中,红细胞裂解法进行样本预富集是一种简单、经济、高效的方法。
Objective To investigate the effect of different pre-enrichment methods on flow cytometry cell sorting of monocyte.Methods Peripheral blood of 15 volunteers were pre-enriched by three methods:erythrocyte lysis,hydroxyethyl starch sedimentation,density gradient centrifugation using Ficoll.Cell viability was detected by Trypan blue staining.The amount and the proportion of monocytes were analyzed by flow cytometry.Results The cell viability of the samples prepared by the3 methods was above 95%.There was no significant difference between the absolute count and grouping of monocytes in erythrocytes lysis and without pre-enrichment samples.The cell subgroup was not obvious by hydroxyethyl starch sedimentation.The number of monocytes obtained by Ficoll density gradient centrifugation was significantly lower than that without pre-enrichment samples.Conclusion Erythrocyte lysis was a high efficient method for monocytes flow cytometry cell sorting.
Objective To investigate the effect of different pre-enrichment methods on flow cytometry cell sorting of monocyte.Methods Peripheral blood of 15 volunteers were pre-enriched by three methods:erythrocyte lysis,hydroxyethyl starch sedimentation,density gradient centrifugation using Ficoll.Cell viability was detected by Trypan blue staining.The amount and the proportion of monocytes were analyzed by flow cytometry.Results The cell viability of the samples prepared by the3 methods was above 95%.There was no significant difference between the absolute count and grouping of monocytes in erythrocytes lysis and without pre-enrichment samples.The cell subgroup was not obvious by hydroxyethyl starch sedimentation.The number of monocytes obtained by Ficoll density gradient centrifugation was significantly lower than that without pre-enrichment samples.Conclusion Erythrocyte lysis was a high efficient method for monocytes flow cytometry cell sorting.
引文
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[2]Ziegler-Heitbrock L.Blood monocytes and their subsets:established features and open questions[J].Frontiers in Immunology,2015(6):423.
[3]李玲,魏大鹏,张平,等.流式细胞术检测外周血白细胞内的磷酸化STAT3[J].现代检验医学杂志,2011,26(3):96-98.Li L,Wei DP,Zhang P,et al.Detecting the tyrosine phosphorylated of STAT3in white blood cells of human peripheral blood by flow cytometry[J].Journal of Modern Laboratory Medicine,2011,26(3):96-98.
[4]Einwallner E,Subasic A,Strasser A,et al.Lysis matters:red cell lysis with FACS lyse affects the flow cytometric enumeration of circulating leukemic blasts[J].Journal of Immunology Methods,2013,390(1/2):127-132.
[5]史艳利,高岭,张宪伟,等.羟乙基淀粉沉降法制备外周血富白细胞血浆的条件优化[J].浙江临床医学,2015,17(7):1204-1205.Shi YL,Gao L,Zhang XW,et al.Optimization of preparation of peripheral blood leukocytes in plasma by hydroxyethyl starch sedimentation[J].Zhejiang Clinical Medical Journal,2015,17(7):1204-1205.
[6]Maqbool M,Vidyadaran S,George E,et al.Optimisation of laboratory procedures for isolating human peripheral blood derived neutrophils[J].Medical Journal of Malaysia,2011,66(4):296-299.
[7]Ji WJ,Lu RY,Liu JX,et al.The influence of different anticoagulants and time-delayed sample processing and measurements on human monocyte subset and monocyte-platelet aggregate analyses[J].Cytometry Part B Clinical Cytometry,2017,92(5):371-379.