Flow Cytometric Analysis of Cell Division by Dilution of CFSE and Related Dyes

A. Bruce Lyons1, Stephen J. Blake2, Kathleen V. Doherty3

1 School of Medicine, The University of Tasmania, Hobart, Tasmania, Australia, 2 Diamantina Institute, The University of Queensland, Brisbane, Queensland, Australia, 3 Faculty of Health Science, The University of Tasmania, Hobart, Tasmania, Australia
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.11
DOI:  10.1002/0471142956.cy0911s64
Online Posting Date:  April, 2013
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


The technique described in this unit uses the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester (CFSE) to track proliferating cells. Covalently bound CFSE is divided equally between daughter cells, allowing discrimination of successive rounds of cell division. The technique is applicable to in vitro cell division, as well as to in vivo division of adoptively transferred cells and can resolve eight or more successive generations. CFSE is long lived, permitting analysis for several months after cell transfer, and has the same spectral characteristics as fluorescein, so monoclonal antibodies conjugated to phycoerythrin or other compatible fluorochromes may be used to immunophenotype the dividing cells. In addition, information is given on a second‐generation dye, Cell Trace Violet (CTV), excited by 405‐nm blue laser light. CTV is chemically related to CFSE, but allows the 488‐nm line of the Argon laser to be used for other probes. Curr. Protoc. Cytom. 64:9.11.1‐9.11.12. © 2013 by John Wiley & Sons, Inc.

Keywords: flow cytometry; cell division; CFSE; CTV; proliferation; adoptive transfer

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Determination of Cell Division Using Carboxyfluorescein Diacetate Succinimidyl Ester (CFDA‐SE or CFSE)
  • Alternate Protocol 1: Determination of Cell Division Using Cell Trace Violet (CTV)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: Determination of Cell Division Using Carboxyfluorescein Diacetate Succinimidyl Ester (CFDA‐SE or CFSE)

  • Single‐cell suspension of cells of interest (e.g., lymphoid cells, cultured cell line)
  • Phosphate‐buffered saline (PBS; appendix 2A)/0.1% (w/v) BSA
  • 5 mM 5‐(and ‐6)‐carboxyfluorescein diacetate succinimidyl ester (CFDA‐SE or CFSE; see recipe)
  • RPMI 1640/10% (v/v) FBS
  • Culture medium or injection medium appropriate for the experiment
  • Antibodies for immunophenotyping (optional)
  • 37°C incubator
  • Flow cytometer with 488‐nm argon laser, or multi‐laser instrument if fluorochromes not excited at 488 nm are being used
  • Additional reagents and equipment for cell culture and harvesting ( appendix 3B) and for immunophenotyping (unit 6.2)

Alternate Protocol 1: Determination of Cell Division Using Cell Trace Violet (CTV)

  • Cell Trace Violet (CTV; see recipe)
  • Multi‐laser flow cytometer with 405‐nm laser
PDF or HTML at Wiley Online Library


  •   FigureFigure 9.11.1 Decay of CFSE fluorescence intensity of nondivided cells. CFSE‐labeled murine splenic lymphocytes (2 × 107) were injected intravenously. At time intervals shown, spleens were removed and analyzed by flow cytometry. The fluorescence intensity of nondividing cells was determined and expressed as a percentage of starting CFSE fluorescence intensity.
  •   FigureFigure 9.11.2 Linearity of staining with respect to CFSE concentration. Murine splenic lymphocytes were stained using the standard protocol with a series of final CFSE concentrations. Fluorescence intensity (arbitrary units) plotted against CFSE concentration shows that staining is linear with respect to CFSE concentration.
  •   FigureFigure 9.11.3 In vitro proliferation of T cells. Murine splenic T cells purified by flow cytometry were stained with CFSE and cultured alone or in the presence of immobilized anti‐CD3 antibody and anti‐CD28. After 3 days of culture, cells were labeled with PE‐conjugated anti‐CD4 or anti‐CD8 antibodies before analysis. Histograms show CFSE fluorescence of undivided control cells, CD4+ T cells, and CD8+ T cells. Note that CFSE staining in conjunction with immunophenotyping allows comparison of the kinetics of proliferation in different populations.
  •   FigureFigure 9.11.4 In vivo B cell division in absence of T cell division after injection of splenic lymphocytes. Murine splenic lymphocytes (2 × 107) labeled with CFSE were injected intravenously. After 14 days, a cell suspension of the recipient's spleen was labeled with anti‐CD45R‐PE to enable discrimination between B and T cells. Events with green fluorescence above the autofluorescence background were collected, ensuring that only CFSE‐positive events were analyzed (∼0.5% of total events in this experiment). A total of 5000 CFSE‐positive events were collected, and anti‐CD45R staining revealed cell division in the B, but not T, cell compartment.
  •   FigureFigure 9.11.5 In vitro alloresponse (mixed lymphocyte response). Murine splenic lymphocytes from BALB/c mice were stained with CFSE and cultured alone (A) or at a 16:1 ratio with C57/Bl6 irradiated, nonlabeled splenocytes (B). After 7 days culture, cells were stained with anti‐CD45R‐PE, and T cells (CD45R) were analyzed by flow cytometry. Note division of a proportion of T lymphocytes cultured with allogeneic stimulators, and no division when cultured alone. Numbered peaks correspond to successive cell divisions at day 7. The number of events in each peak can be determined, allowing the frequency of responders in the starting population to be calculated (Table ).


Literature Cited

   Akbarian, V., Wang, W., and Audet, J. 2012. Measurement of generation‐dependent proliferation rates and death rates during mouse erythroid progenitor cell differentiation. Cytometry A 81:382‐389.
   Ashley, D.M., Bol, S.J., Waugh, C., and Kannourakis, G. 1993. A novel approach to the measurement of different in vitro leukaemic cell growth parameters: The use of PKH GL fluorescent probes. Leukemia Res. 17:873‐882.
   Ashokkumar, C., Gupta, A., Sun, Q., Higgs, B.W., Ningappa, M., Snyder, S., Johnson, M., Mazariegos, G., Soltys, K., Bond, G., Abu‐Elmagd, K., and Sindhi, R. 2010. Proliferative alloresponse of T cytotoxic cells identifies rejection‐prone children with small bowel transplantation. Transplantation 89:1371‐1377.
   Biegler, B.W., Yan, S.X., Ortega, S.B., Tennakiin, D.K., Racke, M.K., and Karandikar, N.J. 2011. Clonal composition of neuroantigen‐specific CD8+ and CD4+ T‐cells in multiple sclerosis. J. Neuroimmunol. 234:131‐140
   Blake, S.J., Hughes, T.P., and Lyons, A.B. 2012. Drug interaction studies evaluating T‐cell proliferation reveal distinct activity of dasatinib and imatinib in combination with cyclosporine A. Exp. Hematol. 40:612‐621.
   Ciantar, J.P., and Mannering, S.I. 2011. An improved method for growing and analyzing human antigen‐specific CD4+ T‐cell clones. Diabetes Metab. Res. Rev. 27:906‐912.
   Deleyrolle, L.P., Rohaus, M.R., Fortin, J.M., Reynolds, B.A., and Azari, H. 2012. Identification and isolation of slow‐dividing cells in human glioblastoma using carboxy fluorescein succinimidyl ester (CFSE). J. Vis. Exp. 62:3918.
   Fulcher, D.A., Lyons, A.B., Korn, S., Cook, M.C., Koleda, C., Parish, C., Fazekas de St. Groth, B., and Basten, A. 1996. The fate of self‐reactive B‐cells depends primarily on the degree of antigen receptor engagement and availability of T‐cell help. J. Exp. Med. 183:2313‐2328.
   Gett, A.V. and Hodgkin, P.D. 2000. A cellular calculus for signal integration by T cells. Nat. Immunol. 240:75‐244.
   Godfrey, W.R., Krampf, M.R., Taylor, P.A., and Blazar, B.R. 2004. Ex vivo depletion of alloreactive cells based CFSE dye dilution, activation antigen selection, and dendritic cell stimulation. Blood 103:1158‐1165.
   Hasbold, J. and Hodgkin, P.D. 2000. Flow cytometric cell division tracking using nuclei. Cytometry 40:230‐237.
   Hasbold, J., Lyons, A.B., Kehry, M.R., and Hodgkin, P.D. 1998. Cell division number regulates IgG1 and IgE switching of B cells following stimulation by CD40L and IL‐4. Eur. J. Immunol. 28:1040‐1051.
   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.
   Hodgkin, P.D., Lee, J.H., and Lyons, A.B. 1996. B cell differentiation and isotype switching is related to division cycle number. J. Exp. Med. 184:277‐281.
   Kindler, V. and Zubler, R.H. 1997. Memory, but not naive, peripheral blood B lymphocytes differentiate into Ig‐secreting cells after CD40 ligation and costimulation with IL‐4 and the differentiation factors IL‐2, IL‐10, and IL‐3. J. Immunol. 159:2085‐2090.
   Kubbies, M., Goller, B., and Van Bockstaele, D.R. 1992. Improved BrdU‐Hoechst bivariate cell kinetic analysis by helium‐cadmium single laser excitation. Cytometry 13:782‐786.
   Lee, H.Y., Hawkins, E.D., Zand, M.S., Mosmann, T., Wu, H., Hodgkin, P.D., and Perelson, A.S. 2009. Interpreting CFSE obtained division histories of B cells in vitro with Smith‐Martin and cyton models. Bull. Math. Biol. 71:1649‐1670.
   Last'ovicka, J., Budinsky, V., Spisek, R., and Bartunkova, J. 2009. Assessment of lymphocyte proliferation: CFSE kills dividing cells and modulates expression of activation markers. Cell. Immunol. 256:79‐85.
   Lyons, A.B. 1997. Pertussis toxin pretreatment alters the in vivo division behaviour and survival of B lymphocytes, after intravenous transfer. Immunol. Cell Biol. 75:7‐12.
   Lyons, A.B. 1999. Divided we stand: Tracking cell proliferation with carboxyfluoresceindiacetate succinimidyl ester. Immunol. Cell Biol. 75:509‐515.
   Lyons, A.B. 2000. Analysing 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.
   Lyons, A.B., Hasbold, J. and Hodgkin, P.D. 2001. Flow cytometric analysis of cell division history using dilution of carboxyfluoresceindiacetate succinimidyl ester, a stably integrated fluorescent probe. Meth. Cell Biol. 63:375‐398.
   Miao, H., Jin, X., Perelson, A.S., and Wu, H. 2012. Evaluation of multitype mathematical models for CFSE‐labeling experiment data. Bull. Math. Biol. 74:300‐326.
   Morrison, R.N., Lyons, A.B., Nowak, B.F., and Hayball, J.D. 2004. Snapper (Pagrus auratus) leucocyte proliferation is synergistically enhanced by simultaneous stimulation with LPS and PHA. Fish Shellfish Immunol. 16:307‐319.
   Nordon, R.E., Ginsberg, S.S., and Eaves, C.J. 1997. High‐resolution cell division tracking demonstrates the Flt3‐ligand‐dependence of human marrow CD34+CD38– cell production in vitro. Br. J. Haematol. 98:528‐539.
   Parish, C.R,, Glidden, M.H., Quah, B.J., and Warren, H.S. 2009. Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation. Curr. Protoc. Immunol. 84:4.9.1‐4.9.13.
   Quah, B.J. and Parish, C.R. 2012. New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE‐like fluorescent dyes. J. Immunol. Methods 379:1‐14.
   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.
   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.
   Wells, A.D., Gudmundsdottir, H., and Turka, L.A. 1997. Following the fate of individual T cells throughout activation and clonal expansion. Signals from T cell receptor and CD28 differentially regulate the induction and duration of a proliferative response. J. Clin. Invest. 100:3173‐3183.
Internet Resources
  The Web site of Invitrogen, the supplier of CFSE/CFDA‐SE and CTV. This site contains much useful information on the application of the CFSE technique, and information on other alternative dyes, including CTV and the orange‐red emitting SNARF‐1 carboxylic acid, acetate, succinimidyl ester.
  The Web site of Verity Software House. ModFit software is used by many researchers to analyze proliferation data; it is available for both PC and Macintosh computers.
PDF or HTML at Wiley Online Library