Abstract:

BACKGROUND: Currently, the immunological detection method has limitation due to complexity of immunoreactions. It is difficult to evaluate body immune status by a single index. Thus, it is urgent to establish a joint evaluation pattern to monitor the immune status of recipients following transplantation.   
OBJECTIVE: To establish a method of donor specific cytotoxicity assay in vivo for quantitatively detecting the immune status of the recipients, and to explore technical parameter of flow cytometry, conditions for fluorescence staining, number of transfused cells, and to determine the optimal testing times.  
METHODS: C57 and Balb/c splenocytes were prepared for cell suspension, labeled by 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) with different final concentrations. A skin transplantation model was established with the recipients BALB/c mice and the donors C57BL/6 mice. Balb/c mice without skin transplantation served as controls. After intravenous transfusion of the CFSE-labeled donor/recipient splenocytes mixture, the following examinations were carried out at 1, 2, 4, 10 hours and 1, 2, 3, 6 days. A quantitative estimate of the ratio of donor/recipient splenocytes labeled with CFSE in host peripheral blood. The percent specific lysis was calculated using the equation.
RESULTS AND CONCLUSION: The flow cytometry was setting lymphocyte gate, and the CRSE concentration was controlled within 20-fold. The number of infused cells had no effects on results in certain range. It is optimal to detect at 2-4 hours after cell transfusion. The results demonstrate that in vivo cell toxicity test is simple, precise, reliable and well suited for quantitatively detecting the immune status of skin transplant recipients in vivo.