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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References
Parish, C.R. Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol. Cell Biol. 77, 499–508 (1999).
Hasbold, J. et al. Quantitative analysis of lymphocyte differentiation and proliferation in vitro using carboxyfluorescein diacetate succinimidyl ester. Immunol. Cell Biol. 77, 516–522 (1999).
Warren, H.S. Using carboxyfluorescein diacetate succinimidyl ester to monitor human NK cell division: analysis of the effect of activating and inhibitory class I MHC receptors. Immunol. Cell Biol. 77, 544–551 (1999).
Lyons, A.B. Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution. J. Immunol. Methods 243, 147–154 (2000).
Parish, C.R. & Warren, H.S. Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation. Curr. Protoc. Immunol. 4.9, 1–10 (2001).
Lyons, A.B. & Parish, C.R. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171, 131–137 (1994).
Weston, S.A. & Parish, C.R. New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy. J. Immunol. Methods 133, 87–97 (1990).
Marzo, A.L. et al. Tumor-specific CD4+ T cells have a major 'post-licensing' role in CTL mediated anti-tumor immunity. J. Immunol. 165, 6047–6055 (2000).
Jedema, I., van der Werff, N.M., Barge, R.M., Willemze, R. & Falkenburg, J.H. New CFSE-based assay to determine susceptibility to lysis by cytotoxic T cells of leukemic precursor cells within a heterogeneous target cell population. Blood 103, 2677–2682 (2004).
Hermans, I.F. et al. The VITAL assay: a versatile fluorometric technique for assessing CTL- and NKT-mediated cytotoxicity against multiple targets in vitro and in vivo. J. Immunol. Methods 285, 25–40 (2004).
Stambas, J., Doherty, P.C. & Turner, S.J. An in vivo cytotoxicity threshold for influenza A virus-specific effector and memory CD8(+) T cells. J. Immunol. 178, 1285–1292 (2007).
Hodgkin, P.D., Lee, J.H. & Lyons, A.B. B cell differentiation and isotype switching is related to division cycle number. J. Exp. Med. 184, 277–281 (1996).
Gett, A.V. & Hodgkin, P.D. Cell division regulates the T cell cytokine repertoire, revealing a mechanism underlying immune class regulation. Proc. Natl. Acad. Sci. USA 95, 9488–9493 (1998).
Bird, J.J. et al. Helper T cell differentiation is controlled by the cell cycle. Immunity 9, 229–237 (1998).
Fazekas de St Groth, B. Carboxyfluorescein diacetate succinimidyl ester and the virgin lymphocyte: a marriage made in heaven. Immunol. Cell Biol. 77, 530–538 (1999).
Nordon, R.E., Nakamura, M., Ramirez, C. & Odell, R. Analysis of growth kinetics by division tracking. Immunol. Cell Biol. 77, 523–529 (1999).
Gett, A.V. & Hodgkin, P.D. A cellular calculus for signal integration by T cells. Nat. Immunol. 1, 239–244 (2000).
De Boer, R.J., Ganusov, V.V., Milutinovic, D., Hodgkin, P.D. & Perelson, A.S. Estimating lymphocyte division and death rates from CFSE data. Bull. Math. Biol. 68, 1011–1031 (2006).
Callard, R. & Hodgkin, P. Modeling T- and B-cell growth and differentiation. Immunol. Rev. 216, 119–129 (2007).
Hawkins, E. et al. Measuring lymphocyte proliferation, survival and differentiation using CFSE time series data. Nat. Protoc. 2, 2057–2067 (2007).
Ueckert, J.E., Nebe von-Caron, G., Bos, A.P. & ter Steeg, P.F. Flow cytometric analysis of Lactobacillus plantarum to monitor lag times, cell division and injury. Lett. Appl. Microbiol. 25, 295–299 (1997).
Khil, L.Y. et al. Insulin has a limited effect on the cell cycle progression in 3T3 L1 fibroblasts. Mol. Cells 7, 742–748 (1997).
Oostendorp, R.A., Audet, J. & Eaves, C.J. High-resolution tracking of cell division suggests similar cell cycle kinetics of hematopoietic stem cells stimulated in vitro and in vivo. Blood 95, 855–862 (2000).
Sukkar, M.B. et al. 'Proliferative' and 'synthetic' airway smooth muscle cells are overlapping populations. Immunol. Cell Biol. 82, 471–478 (2004).
Macaulay, A.E., DeKruyff, R.H., Goodnow, C.C. & Umetsu, D.T. Antigen-specific B cells preferentially induce CD4+ T cells to produce IL-4. J. Immunol. 158, 4171–4179 (1997).
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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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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DOI: https://doi.org/10.1038/nprot.2007.296
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