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  • PBMC
  • Vybrant® CFDA SE Cell Tracer Kit (Molecular Probes) or similar reagent
  • Antigens
  • PBS or HBSS
  • Culture Medium (X-VIVO™ 15 or Iscove’s Modified Eagle Medium with 2% human AB serum are suggested for culture of human PBMC)

Labeling cells with carboxyfluorescein succinimidyl ester (CFSE) is an elegant technique for measuring cell proliferation. Cell surface proteins are stably labeled and, as cells divide, the fluorescent label is equally divided between daughter cells. This allows a count of the number of rounds of division. It also allows labeling of the proliferating cells with additional markers.


  1. Prepare cell suspension for labeling by determining volume necessary for 107 cells per mL.
  2. Prepare CFDA according to kit instructions. Add 90 ul of DMSO to one vial of CFDA and mix well. This makes a 10 mM solution.
  3. Remove 10 uL and dilute in 10 mL of PBS or HBSS to make a 10 uM solution.
  4. Dilute the 10 uM solution of CFDA 1:20 to make enough 0.5 uM CFDA to resuspend the cells at 107 per mL.
  5. Centrifuge the cells to be labeled for 10 minutes at 200 x g.
  6. Decant the supernatant and resuspend the cell pellet in 0.5 uM CFDA.
  7. Incubate for 15 minutes at 37°C to allow the dye to diffuse into the cells.
  8. Centrifuge the cell suspension, resuspend the pellet in culture medium and incubate for 30 minutes at 37°C.
  9. After incubation, centrifuge the cells once more for 10 minutes at 200 x g.
  10. Decant the supernatant and resuspend the cell pellet in culture medium to a concentration of 5 x 106 per mL.
  11. Add cells to a 24 well plate at 1 mL per well or to a 48 well plate at 0.5 mL per well.
  12. Add an equal volume of the antigen, mitogen or agent to be tested.
  13. Incubate at 37°C in 5% CO2 for 4–7 days. For mitogens such as PHA, 4 days is optimum, but for other antigens a 7-day incubation allows for greater expansion of the activated cells.
  14. At the end of incubation, collect cells and counterstain if desired. CFDA is detected in the FL1 channel, so select viability stains or antibody conjugates that are detected in FL2 or FL3.


  • A well containing no antigen must be included for comparison to antigen-stimulated cells.
  • A portion of cells may be left unlabeled to set background fluorescence or compensation.
  • View sample proliferation data below. Use of this method with T regulatory cells is shown here.


  • Vybrant is a trademark of Molecular Probes, Inc.
  • X-VIVO 15 is a trademark of Lonza

CFSE Data Example

CFSE graphs dataOur experience with CFSE is that polyclonal stimuli such as PHA or anti CD3/anti CD28 will result in distinct generations of T cells, but antigen-specific stimulation shows the stimulated population at one level of division. This is shown in the figure below.

In all plots, the x-axis is CFSE fluorescence and the y-axis is staining with anti CD4 phycoerythrin. The upper left plot is an unstimulated CFSE-labeled culture, the upper right is a culture stimulated with tetanus toxoid, the lower left plot is stimulated with Astarte’s CMV antigen, and the lower right plot shows a polyclonal stimulation with PHA.