微创采血结合多色流式细胞术分析小鼠外周血记忆T细胞亚群

目的 为实现序贯多时间连续采血监测小鼠外周血记忆T细胞亚群的变化情况，建立并优化微创采血结合多色流式细胞术分析的方法体系。方法 经隐静脉进行微创采血，使用荧光补偿微球进行多色荧光补偿矩阵的建立，在BD Calibur双激光流式细胞仪上分析小鼠外周血记忆T细胞亚群（初始T细胞、中心记忆T细胞、效应记忆T细胞），评估采血体积、全血是否离心、抗体孵育浓度、红细胞裂解后是否洗涤等参数对多色流式分析结果的影响。随后采用优化的流式细胞分析方法动态评估C57和动脉粥样硬化（AS）易发载脂蛋白E基因敲除（apoE^-/-）小鼠在从常规饮食切换至高脂饮食过程中记忆T细胞的演变过程。结果 （1）经小鼠隐静脉可实现10～50 μL采血，该采血可无需对实验动物进行麻醉，能够实现多次重复采血，且10 μL全血即可满足多色流式细胞术分析的需要，并减少抗体用量。（2）联合高速离心分离血细胞进一步缩小抗体孵育体积，则可进一步节省抗体的用量。（3）对抗体浓度的优化能在保证检测结果准确性和可重复性好的基础上进一步降低抗体用量和背景荧光的干扰。（4）红细胞裂解后加用洗涤可进一步降低背景荧光，但延长操作时间；也可裂解后直接上机检测。（5）使用上述流式细胞学分析方法揭示：apoE^-/-小鼠在常规饮食基线水平其体内的效应记忆T细胞水平即高于对照C57小鼠（P<0.05），给予高脂饮食3周后基本达到平台，而C57小鼠在高脂饮食2周即达到平台，表明apoE^-/-小鼠在AS斑块进展早期即出现免疫调节紊乱。结论 采用隐静脉采血结合优化流式细胞术抗体孵育流程可实现序贯连续采血，完成对小鼠体内记忆T细胞亚群的动态观察。

Establishment and optimization of multi-color flow cytometry analysis of memory T cell subsets in mouse peripheral blood with minimally invasive blood sample collection technique

Objective In order to monitor the dynamic change of memory T cell subsets in mouse peripheral blood, an optimal protocol with combination of minimally invasive blood sample technique and flow cytometry analysis was established. Methods The blood was sample via saphenous vein, and the four color compensation matrix of flow cytometry was established with fluorescence compensation beads, then the ratios of naive T cells, central memory T cells and effector memory T cells among blood samples were analyzed using BD Calibur equipped with two laser. The following aspects were investigated to optimize the flow cytometry protocol:(1) blood sampling volume; (2) centrifugation of blood or not; (3) the concentration of detecting antibodies; (4) wash after the lysis of erythrocyte or not. Results (1) Ten to fifty microliter of blood could be sampled via saphenous vein without animal anesthesia, which would satisfy the requirements of serial blood sampling and analysis, ten microliter blood could be fulfill the requirements of multi-color flow cytometry analysis and decrease the demand of antibodies. (2) Demand of antibodies could be used to detect decreased blood after high speed centrifugation and removal of serum or plasma. (3) Optimization of antibody concentration would decrease the volume of antibodies and potential interference of the background fluorescence without influencing the accuracy and reproducibility. (4) Wash after the lysis of erythrocyte could furthermore decrease the background fluorescence of samples, but increase the operation time, which could be analyzed without wash immediately. Conclusion Combination of blood sampling via saphenous vein and optimal flow cytometry analysis protocol could help to monitor the change of memory T cell subsets in vivo of mouse in a serial of time-points.