Molecular Probes' Vybrant CFDA SE Cell Tracer Kit

The measurement of cell proliferation and turnover, important in many aspects of cell biology, is generally limited by methodological problems. Measuring tritiated thymidine incorporation into DNA is the most commonly used approach for determining cell division, however this method is not appropriate for the analysis of cell division at the level of the individual cell. Conversely, techniques using the nucleotide analogue BrDU do not allow one to make a distinction between progeny of cells that have undergone a single round of division versus those that have undergone several rounds. The fluorescein-derived dye CFDA SE overcomes these problems - it not only allows one to perform long-term cell tracing studies but it also provides a simple and sensitive tool for the analysis of the division history of individual cells.

CFDA SE (also called CFSE) is a non-fluorescent dye that passively diffuses into the cytoplasm of cells. Once inside the cell, it is cleaved by intracellular esterases, becoming fluorescent. This highly amine-reactive product then forms dye-protein adducts that are retained by the cells throughout their development. Moreover CFSE is inherited equally by daughter cells after division, resulting in the sequential halving of mean fluorescence with each generation. When analyzed by flow cytometry, this sequential halving of fluorescence is visualized as distinct peaks and can be used to track division progression. Applications include imaging cultured cells by fluorescence microscopy and analysis of cell division by flow cytometry for in vitro studies as well as for cells adoptively transferred in vivo.

I currently use the Vybrant CFDA SE Cell Tracer Kit for the analysis of antigen-specific and non-specific T-cell proliferation, both in vitro and in vivo after injection into a host animal. The cell tracer kit contains high quality DMSO for the preparation of the stock solution. Even if Molecular Probes says that this stock solution should be prepared immediately prior to use, I currently conserve aliquots at –20C and dilute them to the appropriate concentration just before labeling cells. The range of concentration depends upon the application and the rate of division of the cells of interest. An incubation time of only 10-15 min at 37C is sufficient to label cells whether adherent or in suspension. I use the CFSE dye in combination with PE- and APC-conjugated monoclonal antibodies for multiple parameter analysis by flow cytometry. In my experience, the limit of detection for T lymphocytes is 8-9 cycles of divisions, due to the compression of peaks as the CFSE intensity approaches autofluorescence levels.

The Vybrant CFDA SE Cell Tracer Kit is easy-to-use and it generally yields very bright, stable and homogeneous labeling. Moreover, the dye is not transferred to adjacent cells after injection or in vitro, which is critical. It is important to note, however, that because of the high fluorescence intensity (FL1) the compensation needs to be set up carefully when used for multiple color flow cytometry analysis.

Corinne Ploix, PhD
Post-Doctoral Fellow
The Scripps Research Institute, San Diego