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CFSE Labeling

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Biological and Medical Sciences > Classification by Description > Reagents, Kits and Assays > Assays > Immunoassays

Editor:  Chris Parish
Branch: Protocol-Wiki
Type: Protocol
Intention: For Publication
Read/Write Permissions: Open Access
Authors :
25.06.2016 - Christopher Dyer  -  Sci-Mate   
08.04.2010 - Chris Parish  -  ANU   
19.03.2009 - Chris Parish  -  ANU   

CFSE labeling infuses cells with a dye that is diluted during successive rounds of cell division.  Facs analysis of CFSE labeled cells provides an assay to identify the number of cell divisions that cell populations under go following stimulation- for up to 8 generations.


[edit] Protocol- High Cell Numbers

  1. Prepare lymphocytes at a concentration of 50 x 106/ml in PBS.
  2. Dilute the stock 5mM CFSE solution 1/100 in PBS (to give a 50µM solution). Add 110µl of this solution per ml of cells (to give a final concentration of 5µM), and mix rapidly.
  3. After 5 minutes at room temperature add 10 volumes of PBS containing 5% FCS, centrifuge the cells and wash 3 times in PBS/FCS.

[edit] Modification- Mouse Lymphocytes

An alternative labeling procedure for mouse lymphocytes is to resuspend cells in Hanks BSS at 100 x 106/ml, add an equal volume of CFSE prediluted to 10µM in BSS while vortexing, and incubate for 10 minutes at 37oC. The cells are then diluted in medium containing 10% FCS and washed.

[edit] Modification- Low cell numbers or cultured cells:

Lymphocytes at concentrations as low as 0.5 x 106/ml can be labeled with CFSE, but it is absolutely essential that there is protein present to buffer the toxic effects of CFSE.


  1. Resuspend freshly isolated lymphocytes at concentrations from 0.5 x 106/ml to 10 x 106/ml in PBS containing 5% FCS or PBS containing 0.1% BSA; OR
  2. Cultured lymphocytes that are quiescent at the end of primary culture are labeled directly in their culture medium (containing 10% FCS) after equilibrating to room temperature.

Then, CFSE is added to a final concentration of 5uM as described above, and the cells washed after 5 minutes.

[edit] Technical notes

  1. Labeling with CFSE occurs rapidly, and it is essential that CFSE is dispersed as evenly and quickly as possible so that labelling of cells is uniform. One strategy to achieve this is to add the cell suspension into the bottom of a 10 ml plastic tube, then whilst holding the tube almost horizontal add the CFSE solution to a non-wetted portion of the plastic at the top of the tube. The tube is then capped whilst still in the near horizontal position, and then rapidly inverted several times to mix the lymphocytes and CFSE solution. An alternate strategy is to predilute the CFSE to 10uM and add an equal volume to the cell suspension while vortexing.
  2. When labeling cultured lymphocytes it is best to add CFSE directly into the existing culture medium without prior centrifugation. When cultured cells are centrifuged they form small aggregates such that individual cells are not exposed equally to CFSE. After labelling cultured lymphocytes with CFSE, the cells are washed in PBS and then incubated for 5 minutes in 0.5mM EDTA to dissociate any aggregates, and washed once more in PBS before resuspending in culture medium for restimulation in culture.
  3. CFSE staining of lymphocytes cannot be measured directly after labeling because of the extremely high fluorescence. The majority of CFSE initially taken up by the cells is not stably incorporated and is lost within the first few days.
  4. Uniform labeling with CFSE is essential to obtain clearly defined peaks following division. That is, the coefficient of variation (CV) of the CFSE-labeled control unstimulated lymphocytes must be small. This is in part determined by the technique for mixing CFSE with the cells, detailed above, and by the uniformity in cell volume of the population.

Adapted from Parish, CR and Warren, HS. Use of the Intracellular Fluorescent Dye 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) to Monitor Lymphocyte Migration & Proliferation. Current Protocols in Immunology, Unit 4.9, Supplement 42, 2001 (Wiley and Sons)

[edit] Key References on the CFSE procedure

Lyons, A.B., and Parish, C.R. 1994. Determination of lymphocyte division by flow cytometry. J. Immunol. Meth. 171:131-137.
This is the original paper describing the use of CFSE to monitor lymphocyte division.

Parish, C.R. 1999. Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol. Cell Biol. 77:499-508.
This paper reviews a range of fluorescent dyes for their use in lymphocyte migration and analysis of cell division.

Gett, A.V., and Hodgkin, P.D. 2000. A cellular calculus for signal integration by T cells. Nature Immunol. 1:239-244.
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.

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