This protocol outlines the carboxyfluorescein diacetate succinimidyl ester (CFSE) method for following the proliferation of human lymphocytes in vitro and mouse lymphocytes both in vitro and in vivo. The method relies on the ability of CFSE to covalently label long-lived intracellular molecules with the highly fluorescent dye, carboxyfluorescein. Following each cell division, the equal distribution of these fluorescent molecules to progeny cells results in a halving of the fluorescence of daughter cells. The CFSE labeling protocol described, which typically takes <1 h to perform, allows the detection of up to eight cell divisions before CFSE fluorescence is decreased to the background fluorescence of unlabeled cells. Protocols are outlined for labeling large and small numbers of human and mouse lymphocytes, labeling conditions being identified that minimize CFSE toxicity but maximize the number of cell divisions detected. An important feature of the technique is that division-dependent changes in the expression of cell-surface markers and intracellular proteins are easily quantified by flow cytometry.

The use of carboxyfluorescein diacetate succinimidyl ester (CFSE) to measure lymphocyte proliferation by flow cytometry has become one of the most widely utilised assays for assessing lymphocyte responses. The properties of CFSE make it ideal for such a task, covalently labelling cells with a long-lived fluorescence of high intensity and low variance with minimal cell toxicity. No dye in the last 20 years has been capable of replicating CFSE in these respects. However, currently CFSE is limited to following a maximum of 7 cell divisions and is not compatible for use with ubiquitously available fluorescein conjugates or other fluorescent molecules with spectral properties similar to fluorescein, such as EGFP. Here we characterise two new fluorescent dyes for measuring lymphocyte proliferation, Cell Trace Violet (CTV) and Cell Proliferation Dye eFluor 670 (CPD), which have different excitation and emission spectra to CFSE and, consequently, are compatible with fluorescein conjugates. We found that while both CTV and CPD can label cells to a high fluorescence intensity, which is long-lived and has low variability and low toxicity and makes them ideal for long-term tracking of non-dividing lymphocytes in vivo, CTV offers possibly the best available alternative to CFSE in the analysis of cell divisions. We also describe how intercellular dye transfer and cell autofluorescence can affect division resolution with the three different dyes and describe labelling conditions for the three dyes that produce ultra-bright lymphocytes for in vivo tracking studies and allow up to 11 cell divisions to be detected when using CFSE and CTV as the fluorescent dyes.

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division [1][2][3] . CFSE covalently labels longlived intracellular molecules with the fluorescent dye, carboxyfluorescein. Thus, when a CFSE-labeled cell divides, its progeny are endowed with half the number of carboxyfluorescein-tagged molecules and thus each cell division can be assessed by measuring the corresponding decrease in cell fluorescence via Flow cytometry. The capacity of CFSE to label lymphocyte populations with a high fluorescent intensity of exceptionally low variance, coupled with its low cell toxicity, make it an ideal dye to measure cell division. Since it is a fluorescein-based dye it is also compatible with a broad range of other fluorochromes making it applicable to multi-color flow cytometry. This article describes the procedures typically used for labeling mouse lymphocytes for the purpose of monitoring up to 8 cell divisions. These labeled cells can be used both for in vitro and in vivo studies. Video LinkThe video component of this article can be found at https://www.jove.com/video/2259/ Protocol 1) Reagent Setup 1.1) CFSE stock solutionThe CFSE dye is purchased as carboxyfluorescein diacetate succinimidyl ester (CFDA, SE) (MW 557.47 g/mole), typically in 25 mg vials. Dissolve the 25 mg in 8.96 mL DMSO for a final stock solution of 5 mM (store as 50-100 μL aliquots at -20 °C for several months). CFDA, SE will react with aqueous solution so it is critical that such exposure be avoided during storage.

The stable incorporation of the intracellular fluorescent dye 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) into cells provides a powerful tool to monitor cell migration, and to quantify cell division, because of the sequential decrease in fluorescent labeling in daughter cells. CFSE-labeled lymphocytes have been used to analyze the relationship between cell division and differentiation of cell function, and cell proliferation versus apoptosis, both in vivo and in vitro, and have allowed analysis of the site of response to antigens in vivo.

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