流式细胞仪分选人外周血T淋巴细胞的方法建立与评价

目的:利用流式细胞仪同时分离外人周血单个核细胞中T淋巴细胞并检测其分离纯度及存活率。方法:本文采用流式细胞仪同时分选人外周血CD4~+、CD8~+T淋巴细胞为例,推而广之,采用人外周血淋巴细胞分离液梯度离心法制备外周血单个核细胞,采用流式细胞仪同时分选CD4~+、CD8~+T淋巴细胞,分离细胞再通过流式细胞仪回测其分离纯度并通过台盼蓝染色检测分离细胞的存活率。结果:采用此方法能有效人外周血细胞CD4~+、CD8~+T淋巴细胞,分选前CD4~+淋巴细胞纯度为(50.5±11.5)%、CD8~+T淋巴细胞纯度为纯度为(15.4±7.1)%;分选后CD4~+T淋巴细胞纯度为(94.3±1.3)%、CD8~+T淋巴细胞纯度为(93.6±1.6)%;分选后CD4~+T淋巴细胞存活率为(95.3±1.8)%,CD8~+T淋巴细胞存活率为(94.8±1.5)%,细胞的形态完整。结论:采用人外周血淋巴细胞分离液梯度离心法制备外周血单个核细胞后利用流式细胞仪分选的方法能够高效、快速的分离人外周血CD4~+、CD8~+T淋巴细胞,且存活率高,为进一步研究其功能提供了保证。采用不同的荧光抗体标记其他淋巴细胞亚群,也能高效、快速的分离出细胞。

Establishment and Evaluation of Flow Cytometry Method for Isolation of T Lymphocytes from Peripheral Blood Mononuclear Cells

ABSTRACT Objective: To establish a method for isolation ofT lymphocytes from peripheral blood mononuclear cells(PBMCs) by flow cytometry, and evaluate the isolation rate and survival rate. Methods: PBMCs were prepared using the human peripheral blood lym- phocyte separation liquid based on gradient centrifugation. CD4⁺, CD8⁺T lymphocytes were sorting from PBMCs by flow cytometry, and the purity of these isolated cells was measured by flow cytometry and the survival rate of these cells was detected using trypan blue stain- ing. Results: By this method, we effectively separated human peripheral blood CD4⁺ and CD8⁺T lymphocytes. Without sorting by flow cytometry, the purity of CD4⁺ lymphocytes is (50.5±11.5)%, and the purity of CD8⁺T lymphocytes is (15.4±7.1)%. Sorting by flow cy- tometry dramatically increased the purity of CD4⁺ and CD8⁺T to (94.3±1.3)%, and 93.6+1.6%, respectively. The survival rate was (95.3±1.8%) for the sorted CD4⁺T lymphocyte, and (94.8±1.5%) for the CD8⁺T lymphocyte. Conclusion: This study demonstrated that flow cytometry is highly efficient in the isolation of CD4⁺ and CD8⁺T lymphocytes from PBMCs, providing a guarantee for further study using these cells. Other T lymphocytes subsets also can be highly efficient isolated from PBMCs when it were labeled with different fluorescent antibodies.