Abstract:

BACKGROUND: Many methods have been used in tracing living cells in vivo, but it remains immature to detect cells using difference concentration of fluorochrome.
OBJECTIVE: To establish an assay method for detecting mouse spleen lymphocytes traced by difference concentration of carboxyfluorescein diacetate succinimidyl ester (CFSE) in vivo.
METHODS: Spleen lymphocytes were isolated from C57BL/6J and BALB/c mice and prepared for cell suspension, followed by mixing with difference concentration of CFSE, and then the cell actively was identified by trypan blue staining. The labeled cells were transferred into BALB/c mice. Flow cytometry were performed at different time points to detect the proportion of positive cells.
RESULTS AND CONCLUSION: The CFSE-labeled spleen lymphocytes were highly expressed green fluoresce with a positive labeling rate of 95%. Column diagram showed 2 independent peaks when the concentration of CFSE had 20-fold difference, namely, compared between 0.3 μmol/L and 6 μmol/L. The heterogenic C57BL/6J splenocytes disappeared at 1 day after transfusion, but the syngeneic BALB/c splenocytes could survive for a long time in the peripheral blood of BALB/c mice. The fluorescence intensity was not decreased in two kinds of splenocytes. The results demonstrated that CFSE-labeling with difference concentration is reliable, stable and convenient for spleen lymphocytes tracing in vivo.