Monitoring cell proliferation in vitro with different cellular fluorescent dyes

Abstract

There are few methods for quantifying cell proliferation. Those tests describe the proliferation kinetics of a cell population, but they do not report the history of single cells, the number and frequency of cell divisions, or the precursor cell frequency. Cell-tracking assays based on dilution of the green-fluorescent protein labelling dye, CFSE, has become the standard for monitoring cell proliferation. Other labelling dyes, e.g. CellTrace Violet and CellVue Claret, are also used for the same purpose. This study aimed to compare these three cell labelling methods for analysing the kinetics of cell viability, proliferation, and precursor cell frequency. Human peripheral blood mononuclear cells stimulated with Concanavalin A (ConA) were used as a model system. After labelling with a cell-tracking dye cells were divided into groups with and without ConA stimulation. From the 5 th to 8 th day, cells were collected and analysed with flow cytometry. Cell viability was not significantly different between labelled and unlabelled cells that received ConA stimulation. The proliferative fraction, proliferation index, and nonproliferative fraction were not significantly different among lymphocytes labelled with different dyes. Precursor cell frequency was also similar among cells labelled with the three cell-tracing dyes. The practical conclusion from our observations is that the results from cells labelled with different tracers may be compared directly and discussed jointly.

Coordinated d-cyclin/Foxd1 activation drives mitogenic activity of the Sonic Hedgehog signaling pathway

“…As opposed to WT iMEFs, no change in cell number was observed following SHH stimulation in Gli2 −/− 3 −/− iMEFs. The mitogenic activity of SHH ligand was further examined using CellTrace™ proliferation dye [21]. Relative to vehicle treatment, SHH stimulation of WT iMEFs caused a leftward shift in fluorescent intensity, indicative of increased cell division (Fig.…”

Appraising the suitability of succinimidyl and lipophilic fluorescent dyes to track proliferation in non-quiescent cells by dye dilution

“…We would argue that if the peak resolution quality is insufficient to accurately determine generational position, then it is simpler and more appropriate to perform a more qualitative appraisal of the data whereby a gate can be set to include all divided cells [30] or use another one of the well described qualitative measures [28]. Often, when division metrics are modelled from poorly resolved proliferation data, it is done on the basis of comparing the number and width of peaks to that of another profile obtained by labelling the same cell type with a better dye and activating the cells under the same conditions and using that to determine how many division should have taken place [31,32]. When we have compared the proliferative profiles of the same cells labelled with different dyes, we have often found either slight or profound differences in the proliferative programme (see CTFR data).…”

Influence of oxygen concentration on T cell proliferation and susceptibility to apoptosis in healthy men and women

“…The dividing cell tracking (DCT) method is one of the cell-tracking assays using green fluorescent protein labelling dye, CFDA-SE which, after conversion into CFSE (carboxyfluorescein succinimidyl ester) inside the cells, allows examining the cell cycle kinetics of T lymphocytes [28]. Apoptotic CD4 + and CD8 + cells were identified as annexin V-positive in the phenotypically distinct populations and additionally in the consecutive generations of dividing lymphocytes visualized by binary diluted CFSE fluorescence [29].…”