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

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.