多聚甲醛处理的昆明小鼠T细胞及其亚群存放不同时间分析  

Analysis of the storage time in paraformaldehyde-fixed T cells and their subsets in Kunming mice

作　　者：郭海婷[1] 胡超[1] 严普 李中原[2] 陈志宝[1] 

机构地区：[1]黑龙江八一农垦大学生命科学技术学院,黑龙江大庆163319 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室,甘肃兰州730046

出　　处：《细胞与分子免疫学杂志》2017年第4期460-464,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(31230073;31172316);甘肃省创新研究群体计划项目(1210RJIA006)

摘　　要：目的确定昆明小鼠CD3e^+ CD4^+ T细胞和CD3e^+ CD8a^+ T细胞两种淋巴细胞的可存放时间。方法无菌收集昆明小鼠抗凝血和脾脏并制备单细胞悬液,经荧光基团标记的抗体对CD3e、CD4、CD8a进行标记、多聚甲醛处理后运用三色标记流式细胞术分点检测4℃避光保存的待检样品的可存放情况,采用最小二乘法拟合最佳回归曲线,计算各时间点上淋巴细胞含量并进行统计学分析,同设多聚甲醛未处理组。结果昆明小鼠脾组织内CD3e^+ CD4^+ T细胞和CD3e^+ CD8a^+ T淋巴细胞经多聚甲醛处理后分别在第11天和第16天显著降低,未经多聚甲醛处理样品的这两个亚群细胞则在第8天和第11天显著降低;经多聚甲醛处理的外周血T淋巴细胞的可存放时间较长(均为第18天),高于未处理组(第10天和第15天)。结论流式细胞术检测多聚甲醛处理的昆明小鼠脾组织和外周血T淋巴细胞应在第11天前完成。Objective To determine the expiration dates of CD3 e＋ CD4 ＋ T and CD3e ＋ CD8a ＋ T lymphocytes of Kunming mice. Methods The sterile spleen and anticoagulant blood of Kunming mice were collected and used to prepare the single-cell suspensions. After CD3e, CD4 and CD8a of T cells were stained with fluorophore-conjugated monoclonal antibodies, the samples were fixed with paraformaldehyde （PFA） and stored at 4℃ in dark followed by detection using three-color flow cytometry at several specific time points. The best regression curves fitted with the least squares method were used for calculating the contents of T cells at each time point and the subsequent statistical analysis. Samples without PFA served as the blank controls. Results The levels of PFA-fixed CD3e ＋ CD4 ＋ T and CD3e ＋ CD8a ＋ T lymphocytes from spleen tissues significantly decreased at 11 days and 16 days post collection, respectively; whereas no difference was found in the samples without PFA until 8 days and 11 days post collection, respectively. As for the T cells separated from the peripheral blood, the longer storage periods were both 18 days under the PFA fixation, which were longer than those of the blank controls （ 10 and 15 days, respectively）. Conclusion The PFA-fixed T lymphocytes of the spleen and peripheral blood of Kunming mice should be detected using flow cytometry within l] days post collection.

关 键 词：昆明小鼠 T淋巴细胞 线性回归 存放时间 流式细胞术 

分 类 号：R392.11[医药卫生—免疫学] N37[医药卫生—基础医学]