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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 shelf life of two lymphocytes from Kunming mouse CD3e ~ + CD4 ~ + T cells and CD3e ~ + CD8a ~ + T cells. Methods Anticoagulant blood and spleen of Kunming mice were collected aseptically and single cell suspension was prepared. CD3e, CD4 and CD8a were labeled with fluorophore-labeled antibody. After treatment with paraformaldehyde, the cells were treated with three-color flow cytometry The stored samples stored at 4 ℃ in darkness were tested. The best regression curve was fit by least square method. The lymphocyte contents at each time point were calculated and statistically analyzed. The same paraformaldehyde untreated group was also prepared. Results After treated with paraformaldehyde, the numbers of CD3e ~ + CD4 ~ + T cells and CD3e ~ + CD8a ~ + T cells in Kunming mice were significantly decreased on the 11th and the 16th days, respectively, and the samples without the paraformaldehyde treatment Of the two subpopulations were significantly reduced on day 8 and day 11; paraformaldehyde-treated peripheral blood T lymphocytes were longer (both on day 18) and higher than untreated ( Day 10 and day 15). Conclusion Flow cytometry of paraformaldehyde-treated Kunming mice spleen tissue and peripheral blood T lymphocytes should be completed by the 11th day.