Application of vital dye CFDA-SE and SNARF-1 to evaluate mixed lymphocyte reaction

Abstract

AIM: To establish a new method for more comprehensive evaluation of responsive intensity in one-way mixed lymphocyte reaction (MLR). METHODS: CFDA-SE labeled lymphocytes from lymph nodes of BALB/cJ mice were applied as responder cells, and SNARF-1 labeled splenocytes from BALB/cJ or C57BL/6 mice which had been pretreated with mitomycin C were applied as stimulator cells to establish mixed lymphocyte culture (MLC). After 96 h of culture, cells were harvested and analyzed by flow cytometry to acquire the proliferative information of SNARF-1 negative and CFSE positive staining responder cells. ModFit software was then used to get more proliferation related index. RESULTS: With increasing division cycles, the CFSE fluorescence was diluted to the extent that the responder cells could not be discriminated from stimulator cells, which was resolved by gating SNARF-1 positive cells out to define responder cells. Several important proliferation related indexes were obtained by ModFit software, such as precursor frequency and proliferation index, etc. CONCLUSION: Our results show that application of vital dye CFDA-SE and SNARF-1, combined with flow cytometry is a powerful tool for evaluation of responsive intensity in one-way MLR.