外周血单个核细胞
离心
差速离心
密度梯度
免疫系统
血细胞
生物
全血
分离(微生物学)
免疫学
色谱法
生物医学工程
化学
分子生物学
医学
生物信息学
体外
生物化学
物理
量子力学
作者
Judith R. Stabel,Taylor L. T. Wherry
标识
DOI:10.1016/j.jim.2022.113407
摘要
Peripheral blood mononuclear cells (PBMCs) are critical for assessment of host immune responses to infectious disease. The isolation of PBMCs from whole blood is a laborious process involving density gradients and multiple centrifugation steps. In the present study we compared a more traditional method of PBMC isolation used in our laboratory to two novel methods of cell isolation for efficiency, cell viability, and enumeration of cell subsets. Our laboratory method uses Histopaque-1077 density gradient in standard conical tubes and this was compared with isolation of cells using SepMate™ tubes, a novel conical tube containing an insert to separate the density gradient. Multiple experiments were performed to optimize the SepMate™ tubes for use with cattle blood. A final experiment was conducted to compare traditional methodology, the optimized SepMate™ method with a more novel method using cell preparation tubes (CPT-10 vacutainers containing density gradient). Results demonstrated that optimization of the SepMate™ tube methodology was necessary, including dilution of blood and addition of centrifugation steps to reduce platelet contamination. The CPT-10 tubes worked well but cell recovery was lower compared to other methods. Both of the newer methods were comparable to a modified version of our traditional laboratory method of PBMC isolation in terms of numbers of recovered viable cells and the frequency of immune cell subsets. Additionally, efficiency was improved, particularly with the SepMate™ tube method, resulting in reduced time in the laboratory as well as reduced usage of plasticware.
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