Use of the Intracellular Fluorescent Dye CFSE to Monitor Lymphocyte Migration and Proliferation

Christopher R. Parish1, Megan H. Glidden1, Ben J. C. Quah1, Hilary S. Warren1

1 Australian National University, Canberra, Australia
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 4.9
DOI:  10.1002/0471142735.im0409s84
Online Posting Date:  February, 2009
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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. Curr. Protoc. Immunol. 84:4.9.1‐4.9.13. © 2009 by John Wiley & Sons, Inc.

Keywords: CFSE; cell division; cell tracking; lymphocyte migration; lymphocyte positioning

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Table of Contents

  • Introduction
  • Basic Protocol 1: CFSE Labeling of Lymphocytes
  • Support Protocol 1: Analysis of CFSE‐Labeled Cells by Flow Cytometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: CFSE Labeling of Lymphocytes

  • Experimental animals or human peripheral blood or cultured lymphocytes
  • Phosphate‐buffered saline (PBS; appendix 2A), pH 7.4
  • Hanks' balanced salt solution (HBSS), pH 7.4 ( appendix 2A)
  • PBS ( appendix 2A) containing 5% (v/v) heat‐inactivated FBS
  • 5 mM CFSE stock solution (see recipe)
  • Antigens and mitogens of interest
  • 0.5 mM disodium EDTA in PBS ( appendix 2A)
  • 1‐ml syringes
  • 25‐G needles
  • Fluorescence microscope with filters for fluorescein
  • Razor blade
  • Additional reagents and equipment for removal of mouse lymphoid organs (unit 1.9), preparation of mononuclear cell suspensions (unit 3.1), and isolation of peripheral blood mononuclear cells (unit 7.1), immunohistochemistry (unit 21.4), culturing mouse (units 3.10& 3.12), or human (units 7.10& 7.11) lymphocytes, and counting cells using a hemacytometer ( 3.NaN)

Support Protocol 1: Analysis of CFSE‐Labeled Cells by Flow Cytometry

  • CFSE‐labeled cells (see protocol 1)
  • Analytical flow cytometer capable of 3‐color fluorescence
  • Cell sorting flow cytometer (for some applications)
  • Additional reagents and equipment for cell surface staining of lymphocytes (unit 5.3), intracellular cytokine staining (unit 6.24), analytical flow cytometry (unit 5.4), flow cytometric analysis of apoptosis (unit 3.17), and cell sorting flow cytometry (unit 5.1)
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Literature Cited

   Ford, W.L. 1978. The preparation and labeling of lymphocytes. In Handbook of Experimental Immunology, Vol. 3 (D.M. Weir, ed.) p. 23.1. Blackwell Scientific, Oxford.
   Garton, H.J. and Schoenwolf, G.C. 1996. Improving the efficacy of fluorescent labeling for histological tracking of cells in early mammalian and avian embryos. Anat. Rec. 244:112‐117.
   Gett, A.V. and Hodgkin, P.D. 2000. A cellular calculus for signal integration by T cells. Nat. Immunol. 1:239‐244.
   Graziano, H.J., St‐Pierre, Y., Beauchemin, C., Desrosiers, M., and Potworowski, E.F. 1998. The fate of thymocytes labeled in vivo with CFSE. Exp. Cell Res. 240:75‐85.
   Hasbold, J. and Hodgkin, P.D. 2000. Flow cytometric cell division tracking using nuclei. Cytometry 40:230‐237.
   Hasbold, J., Gett, A.V., Rush, J.S., Deenick, E., Avery, D., Jun, J., and Hodgkin, P.D. 1999. Quantitative analysis of lymphocyte differentiation and proliferation in vitro using carboxyfluorescein diacetate succinimidyl ester. Immunol. Cell Biol. 77:516‐522.
   Hawkins, E.D., Hommel, M., Turner, M.L., Battye, F.L., Markham, J.F., and Hodgkin, P.D. 2007. Measuring lymphocyte proliferation, survival and differentiation using CFSE time‐series data. Nat. Protoc. 2:2057‐2067.
   Hermans, I.F., Silk, J.D., Yang, J., Palmowski, M.J., Gileadi, U., McCarthy, C., Salio, M., Ronchese, F., and Cerundolo, V. 2004. 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.
   Jedema, I., van der Werff, N.M., Barge, R.M., Willemze, R., and Falkenburg, J.H. 2004. 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.
   Khil, L.Y., Kim, J.Y., Yoon, J.B., Kim, J.M., Keum, W.K., Kim, S.T., Yoon, Y., Yoon, M.Y., Moon, C.K., Lee, J.H., Ha, J., Kim, S.S., and Kang, I. 1997. Insulin has a limited effect on the cell cycle progression in 3T3 L1 fibroblasts. Mol. Cell 7:742‐748.
   Lyons, A.B. 1999. Divided we stand: Tracking cell proliferation with carboxyfluorescein diacetate succinimidyl ester. Immunol. Cell Biol. 77:509‐515.
   Lyons, A.B. 2000. Analyzing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution. J. Immunol. Methods 243:147‐154.
   Lyons, A.B. and Parish, C.R. 1994. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171:131‐137.
   Marzo, A.L., Kinnear, B.F., Lake, R.A., Frelinger, J.J., Collins, E.J., Robinson, B.W., and Scott, B. 2000. Tumor‐specific CD4+ T cells have a major “post‐licensing” role in CTL mediated anti‐tumor immunity. J. Immunol. 16:6047‐6055.
   Nordon, R.E., Nakamura, M., Ramirez, C., and Odell, R. 1999. Analysis of growth kinetics by division tracking. Immunol. Cell Biol. 77:523‐529.
   Oostendorp, R.A., Audet, J., and Eaves, C.J. 2000. 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.
   Parish, C.R. 1999. Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol. Cell Biol. 77:499‐508.
   Quah, B.J., Warren, H.S. and Parish, C.R. 2007. Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester. Nat. Protoc. 2:2049‐2056.
   Stambas, J., Doherty, P.C., and Turner, S.J. 2007. An in vivo cytotoxicity threshold for influenza A virus‐specific effector and memory CD8(+) T cells. J. Immunol. 178:1285‐1292.
   Sukkar, M.B., Stanley, A.J., Blake, A.E., Hodgkin, P.D., Johnson, P.R., Armour, C.L., and Hughes, J.M. 2004. ‘Proliferative’ and ‘synthetic’ airway smooth muscle cells are overlapping populations. Immunol. Cell Biol. 8:471‐478.
   Ueckert, J.E., Nebe von‐Caron, G., Bos, A.P., and ter Steeg, P.F. 1997. Flow cytometric analysis of Lactobacillus plantarum to monitor lag times, cell division and injury. Lett. Appl. Microbiol. 25:295‐299.
   von Horsten, S., Helfritz, A., Kuhlmann, S., Nave, H., Tschernig, T., Pabst, R., Ben‐Eliyahu, S., Meyer, D., Schmidt, R.E., and Schmitz, C. 2000. Stereological quantification of carboxyfluorescein‐labeled rat lung metastasis: A new method for the assessment of natural killer cell activity and tumor adhesion in vivo and in situ. J. Immunol. Methods 239:25‐34.
   Warren, H.S. 1999. 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.
   Warren, H.S. and Kinnear, B.F. 1999. Quantitative analysis of the effect of CD16 ligation on human NK cell proliferation. J. Immunol. 162:735‐742.
   Weston, S.A. and Parish, C.R. 1990. New fluorescent dyes for lymphocyte migration studies: Analysis by flow cytometry and fluorescence microscopy. J. Immunol. Methods 133:87‐97.
Key References
   Gett and Hodgkin, 2000. See above.
  This paper is an elegant demonstration of the power of CFSE to monitor lymphocyte division using a mathematical model for assessing the response of lymphocytes to different signals.
   Lyons and Parish, 1994. See above.
  This is the original paper describing the use of CFSE to monitor lymphocyte division.
   Parish, 1999. See above.
  This paper reviews a range of fluorescent dyes for their use in lymphocyte migration and analysis of cell division.
   Hawkins et al., 2007. See above.
  An overview of the different approaches to analyze CFSE data.
Internet Resources
  ModFit LT software is an excellent software for cell proliferation analysis and is available for Macintosh and PC.
  FACS data analysis software, which is currently available for Macintosh and PC.
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