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Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester

Abstract

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.

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Figure 1: A schematic representation of the various molecular events that occur during the labeling of cells with CFDASE.
Figure 2: Demonstration of the effect of different CFSE labeling conditions on the ability of human PBMCs to respond to the polyclonal T-cell mitogen PHA.
Figure 3: Measurement of CD4+ T-cell proliferation in vitro by CFSE dilution.
Figure 4: Measurement of CD4+ T-cell proliferation in vivo by CFSE dilution.

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Acknowledgements

This work was supported by a National Health and Medical Research Council (NHMRC) of Australia Program Grant (C.R.P.) and NHMRC Project Grants (H.S.W. and B.J.C.Q.). Also B.J.C.Q. is an NHMRC Peter Doherty Postdoctoral Fellow and H.S.W. is an NHMRC Senior Research Fellow.

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Correspondence to Christopher R Parish.

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Quah, B., Warren, H. & Parish, C. Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester. Nat Protoc 2, 2049–2056 (2007). https://doi.org/10.1038/nprot.2007.296

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