Abstract:

This study aimed to optimize the flow cytometry detection method of T lymphocyte subsets in peripheral blood. Heparin sodium anticoagulant blood was collected from mice， cell staining was performed with fluorescent antibodies FITC rat anti-mouse CD3， APC rat anti-mouse CD4 and PE rat anti-mouse CD8a， and red blood cells were lysed. The percentage of each subclass of cells in lymphocytes was determined by flow cytometry. The method was optimized from these aspects， including sample volume， working concentrations of fluorescent antibodies including FITC rat anti-mouse CD3 （1.25~40.00 μg·mL^-1）， APC rat anti-mouse CD4 （0.625~20.000 μg·mL^-1）， PE rat anti-mouse CD8a （1.25~40.00 μg·mL^-1） and the conditions of red blood cells lysis （times and number of lysis）. The precision of the method was verified from intra-batch and inter-batch differences. Meanwhile，the stability of the samples at 4 ℃ for 24 h， at room temperature for 24 h and at 4 ℃ for 24 h after staining was verified. The results showed that the final concentration of FITC rat anti-mouse CD3， APC rat anti-mouse CD4 and PE rat anti-mouse CD8a were 1.25 μg·mL^-1， 1.25 μg·mL^-1 and 5.00 μg·mL^-1 respectively in 50 μL of anticoagulant blood. Red blood cells lysis buffer were added for 5 min after incubation for 30 min at room temperature without light， then phosphate buffer saline （PBS） was added to stop lysing. A repeated lysis was required. Then cells were resuspended in PBS， and detected on the machine. The intra-batch difference and inter-batch difference of the optimized method were both less than 15%， which met the requirements. The stained sample could remain stable after being placed at 4 ℃ for 24 hours. The basic methods of flow cytometry detection of T cell subsets in peripheral blood of mice were optimized， summarized and verified， which provided research methods and experimental data for preclinical immunotoxicity evaluation and provided reference for the study of flow cytometry to analyze the immunophenotype of clinical blood lymphocytes.

Key words: flow cytometry, T lymphocyte subsets in peripheral blood, optimization

摘要：

为优化外周血T淋巴细胞亚群流式检测方法，采集小鼠肝素钠抗凝血，用FITC rat anti-mouse CD3、APC rat anti-mouse CD4和PE rat anti-mouse CD8a荧光抗体进行染色，裂解红细胞，通过流式细胞仪检测各亚类细胞占淋巴细胞百分比。从样本体积（50 μL和100 μL）、FITC rat anti-mouse CD3抗体的工作浓度（1.25~40.00 μg·mL^-1）、APC rat anti-mouse CD4抗体的工作浓度（0.625~20.000 μg·mL^-1）、PE rat anti-mouse CD8a抗体的工作浓度（1.25~40.00 μg·mL^-1）及红细胞裂解条件（时间和裂解次数）等方面对检测方法进行了优化，并验证方法的精密度（批内差异和批间差异）；同时对样品4 ℃放置24 h、室温放置24 h、染色处理后4 ℃放置24 h的稳定性进行验证。结果表明，50 μL抗凝血中加入终浓度1.25 μg·mL^-1 FITC rat anti-mouse CD3、1.25 μg·mL^-1 APC rat anti-mouse CD4和5.00 μg·mL^-1 PE rat anti-mouse CD8a，室温避光孵育30 min，加入红细胞裂解液，裂解5 min，用磷酸盐缓冲液（phosphate buffer saline，PBS）终止裂解后，重复裂解1次，再用PBS重悬后上机检测，批内差异和批间差异均<15%，符合标准。染色处理后样品4 ℃放置24 h可保持稳定性。对流式检测小鼠外周血T细胞亚群的基本方法进行全面优化及验证，旨在为临床前免疫毒性评价提供研究方法和实验数据，以及为流式细胞术分析临床血液淋巴细胞免疫表型的方法学研究提供参考依据。

关键词: 流式细胞术, 外周血T淋巴细胞亚群, 优化