热变性对外周血细胞亚群标记效果的影响

Effect of heat denaturation on labeled peripheral blood cell subsets

目的探讨热变性对外周血细胞亚群标记效果的影响,从而建立稳定的流式-荧光原位杂交技术流程(flow cytometryfluorescence in situ hybridization,Flow-FISH)。方法采集10例健康人肝素抗凝新鲜外周血标本,分别以Alexa Fluor647标记的CD3、CD19、CD14抗体标记T淋巴细胞、B淋巴细胞和单核细胞,高温变性后,采用流式细胞术分析热变性前后细胞系列特异性抗原表达的变化;以Fluor647-CD3标记外周血T淋巴细胞,以4种浓度的Bis[sulfosuccinimidyl]suberate(BS3)交联剂进行抗原抗体交联,比较不同浓度BS3的交联效果。结果高温变性后,Alexa Fluor647标记的CD3和CD19的荧光强度减弱,但仍能有效区分出T淋巴细胞和B淋巴细胞;Alexa Fluor647-CD14的荧光强度无变化,单核细胞清晰可辨;4种浓度交联剂BS3作用后,CD3的标记效果基本一致。结论经过热变性后,Alexa Fluor647标记的CD3、CD19、CD14抗体仍能够有效标记出血细胞中的不同细胞亚群,耐热性好,可以用于Flow-FISH技术中;交联剂BS3的适宜作用浓度为2.5 mmol/L。

Objective To investigate the effect of heat denaturation on labeled peripheral blood cell subsets and establish a stable protocol of flow cytometry-Fluorescence in situ hybridization（ Flow-FISH）. Methods Heparinized fresh peripheral blood samples were taken from ten healthy persons. T lymphocytes,B lymphocytes and monocytes were labeled with Alexa Fluor647-CD3,CD19 and CD14 seperately,then denatured by high temperature. The expression of lineage-specific antigens were detected by flow cytometry,on cells with and without heat denaturation separately. T lymphocytes were labeled with Alexa Fluor647-CD3,and were allowed to react with four concentrations of crosslinker Bis[sulfosuccinimidyl] suberate（ BS3） seperately,and the efficiency was evaluated by flow cytometry.Results After heat denaturation,the expression of CD3 and CD19 became weaker,while T lymphocytes and B lymphocytes could still be differentiated effectively; the expression of CD14 did not change,and monocytes could be differentiated clearly. After the reaction with BS3 in four concentrations,the percentages of Alexa Fluor647-CD3 labeled T lymphocytes were similar. The application of Alexa Fluor647 labeled anti-CD3,CD19 and CD14 antibodies might make the differentiation of blood cell subsets by Flow-FISH feasible,and the optimal final concentration of BS3 would be 2. 5 mmol /L.