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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

1Australian 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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Christopher Parish

Abstract

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: CFSE Labeling of Lymphocytes
  • Support Protocol: Analysis of CFSE-Labeled Cells by Flow Cytometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Other Versions
     
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Materials

Basic Protocol: CFSE Labeling of Lymphocytes

 Materials
  • 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 (appendix 3A)

Support Protocol: Analysis of CFSE-Labeled Cells by Flow Cytometry

 Materials
  • CFSE-labeled cells (see Basic Protocol)
  • 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)
Looking for materials?  Suppliers Guide
     
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Figures

  • Figure 4.9.1
    Progressive gating strategy for detection of maximum T cell divisions in vivo. OVA-specific V2+, CD8+ T cells were purified from OT-I mice (bred on a CD45.1 background) and labeled with CFSE. T cells (5 × 106) were then intravenously injected into C57BL/6J mice (which have a CD45.2 background). At a time point 2 hr later, mice were intravenously injected with either 2, 20, or 200 µg of OVA (in PBS) or PBS alone as a control. After 3 days, splenocytes were harvested, labeled with Hoechst 33258 (at 1 µg/ml for dead cell discrimination) and APC-conjugated anti-CD8, PerCP-Cy5.5-conjugated anti-B220, PE-conjugated anti-V2, and PE-Cy7 conjugated anti-CD45.1 monoclonal antibodies, and 1 × 106 cells were collected on an LSR-II (BD biosciences) for analysis. (A) Progressive gating on subpopulations of splenocytes reveals higher resolution of CFSE profiles of transferred cells (with CD8+, CD45.1+ viable cells giving the highest resolution). (B) Detection of division of transferred CFSE-labeled OT-I CD8+ T cells to increasing amounts of antigen (OVA).

    View Image
  • Figure 4.9.2
    CFSE profiles for PBMC labeled at 1 × 106 /ml in PBS containing 5% FBS with 5 µm CFSE for 5 min and cultured with 5 µM phytohemagglutinin for 4 days. The unfilled peak on the right at 7.6 × 106 fluorescence units corresponds to the control unstimulated CFSE-labeled cells. The unfilled peak on the left at 2 × 100 fluorescence units corresponds to the autofluorescence of unlabeled cells. The calculated marker positions are shown: M1 (undivided), M2 (1 division), M3 (2 divisions), M4 (3 divisions), M5 (4 divisions), M6 (5 divisions), and M7 (6 divisions).

    View Image
  • Figure 4.9.3
    A schematic representation of the various stages in the labeling of cells with carboxyfluorescein diacetate succinimidyl ester. For details see text. The size of the arrows in the figure is proportional to the rate of diffusion of the different molecules through the cell membrane. The carbonyl group located between positions 5 and 6 on the benzene ring is to indicate that the dye is a mixture of 5- and 6-carbonyl structural isomers. Figure reproduced from Parish (1999) with kind permission of the publishers (Blackwell Science Asia).

    View Image
  • Figure 4.9.4
    The relationship between the geometric mean fluorescence of CFSE in undivided human PBMC and the response of CFSE-labeled PBMC to 5 µM phytohemagglutinin, measured as the percentage of cells that have entered division after 4 days culture. Data were calculated as described in Figure 4.9.2.

    View Image

Literature Cited

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
    http://www.vsh.com/

ModFit LT software is an excellent software for cell proliferation analysis and is available for Macintosh and PC.

    http://www.flowjo.com/

FACS data analysis software, which is currently available for Macintosh and PC.

     
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