Minimally invasive blood sample collection combined with multi-color flow cytometry for analyzing memory T cell subsets in mouse peripheral blood / 第二军医大学学报

ABSTRACT

Objective: To establish an optimized method by combining minimally invasive blood sample technique and flow cytometry analysis for monitoring the dynamic changes of memory T cell subsets in mouse peripheral blood. Methods: The blood samples were collected via the 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 were analyzed using BD Calibur equipped with two laser. The following factors were investigated to optimize the flow cytometry protocol (1) blood sampling volume; (2) centrifugation of blood or not; (3) the concentration of detecting antibodies; and (4) whether to wash after the lysis of erythrocytes. The optimized protocol was used to investigate the dynamic changes of memory T cells in C57 and apoE knockout (apoE-/-) mice during switching from chow to high fat diet. Results: (1) 10-50 μL blood samples could be collected via the saphenous vein of mice without anesthesia, and the process could be repeated and 10 μL blood sample could meet the requirement for multi-color flow cytometry analysis, which reducing the demand of antibodies. (2) Demand of antibodies could be further reduced by high speed centrifugation and removal of serum or plasma. (3) Optimization of antibody concentration could further reduce the amount of antibodies and potential interference of the background fluorescence without influencing the accuracy and reproducibility. (4) Washing after the lysis of erythrocyte could further decrease the background fluorescence of samples, but it increased the operation time, and it could also be analyzed without washing. (3) The effector memory T cell level of apoE--/- mice was significantly higher than that of C57 mice at the baseline level of chow diet (PC<.05); the level increased to a plateau after 3-week high fat diet in apoK-/- mice and after 2-week high fat diet in C37 mice, indicating immunity dysfunction during early stages of atherosclerosis in apoE -/- mice. Conclusion: Combination of blood sampling via saphenous vein and optimal flow cytometry analysis protocol can help to monitor the dynamic change of memory T cell subsets in vivo in mice.