Tracking cell proliferation using the far red fluorescent dye SNARF‐1

Abstract

The [(3)H]thymidine incorporation assay and staining of living cells with fluorescent dyes like carboxyfluorescein diacetate, succinimidyl ester (CFSE) have evolved as valuable methods for studying T cell responses. To assess proliferation of cells already labeled by FITC, CFSE, GFP, or other "green" molecules or to simultaneously track two otherwise indistinguishable cell populations in mixed cell cultures, it would be desirable to have a dye with distinct fluorescent properties for this application. We analyzed the dilution of the far red fluorescent dye SNARF-1 in proliferating cells by flow cytometric analysis. The results were compared with the CFSE dilution technique as well as the [(3)H]thymidine incorporation assay. Staining of primary human lymphocytes revealed that SNARF-1 labeling was equivalent to CFSE for estimating proportions of proliferating cells in stimulated cell cultures and yielded results comparable to [(3)H]thymidine incorporation. We showed that SNARF-1 offers the possibility to simultaneously analyze the proliferation of phenotypically indistinguishable subsets of hematopoietic cells and can also be used to track uniformly proliferating, non hematopoietic cells like HEK293. In summary, we have demonstrated that labeling of cells with SNARF-1 allows for estimating cell proliferation of cells of hematopoietic and non-hematopoietic origin.