Savina Stoitsova

CFSE Staining: a new way to look at cell division

CFSE = Carboxyfluorescein diacetate,succinimidyl ester.

CFSE is able to irreversibly couple tointracellular and cell-surface proteinsthrough a reaction with available aminegroups (for example, on lysine sidechains). Its irreversible association withlow-turnover proteins (like cytoskeletalproteins) commits CFSE to stay in thecells through a series of cell divisions,where the amount of CFSE in daughtercells decreases twofold after eachdivision.

Figure 1: Chemical formula of CFSE.Figure reproduced from:http://www.dojindo.com/newprod/1/cell-v/staining/89gif/cfse.gif

The Chemistry Behind

CFSE is a fluorescein derivative, with twoacetate and one succinimidyl ester groups. Inthis form it is membrane permeant and non-fluorescent. When added into a cellularsuspension it thus diffuses easily into cells. Inthe cytoplasm, endogenous esterases cleaveoff the acetate groups, making the moleculefluorescent and unable to pass the membraneto the outside. The succinimidyl ester thenreacts with free amine groups of cellularproteins.Random binding to amine groups might affectthe activity of proteins. This is why one hasto be careful with the amounts of CFSEapplied to a cellular population. The optimalamounts vary according to the types of cellsbeing monitored.Initially, after incubation the generalfluorescence of CFSE drops rapidly due todegradation of high-turnover proteins. Lateron the level stabilizes, and CFSE can remainfor a long time bound to low-turnoverproteins. The stability of CFSE makes it quitedurable: six months after incubation,

Figure 2: Proliferating B cells stainedwith CFSE and analyzed by FACS.From Hodgkin, P.D., Lee, J.H., Lyons, A.B., 1996.J. Exp. Med. 184, 277–281 (Lyons A.B, 2000)

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undivided cells retain about 10% of their fluorescence. In this respect CFSE is suitablefor long-term experiments.

Common Applications of CFSE

Labeling of cells with CFSE has allowed for detailed monitoring of subpopulations ofcells within one bulk population. Because fluorescence intensity is reduced exactlytwofold with each division, one can separate through FACS subsets of cells that havestopped dividing from ones that are still dividing (see figure 2), and also visualize thedivision cycle at which a particular subset has stopped dividing.Combining the method with other labeling procedures, compatible with CFSE, one canalso monitor the differentiation patterns of cells, according to changes in their surfacemarkers, immunophenotype, cytokine expression, DNA content and other factors. In thisway CFSE labeling has become a very useful tool in the differentiation analysis of cellsof the immune system.

Figure 3: CFSE labeling combinedwith labeling for different markers.The vertical bars across the plots markeach division cycle, where CFSEfluorescence decreases with eachdivision (X-axis).Labeling for CD45R(a), B7.1 (b), B7. (c), sydecan (d), CD23(e) and CD16/32 (f) shows the kineticsof their expression during differentiationin the cellular population

Figure is reproduced from Hodgkin, P.D., Lee, J.H.,Lyons, A.B., 1996. J. Exp. Med. 184, 277–281(Lyons A.B, 2000)

Another Application, as described in the presented article “Toll-dependent selectionof microbial antigens for presentation by dendritic cells” (Blander J. M andMedzhitov R., 2006):

The authors here used CFSE to create a bulk of fluorescent apoptotic B-cells. The B-cells were incubated with CFSE for 15min at 37oC and then their apoptosis was inducedthrough UV radiation. The fluorescent apoptotic cells were incubated with viabledendritic cells. The CFSE fluorescence of the dendritic cells was used to visualize theamount of phagocytosed apoptotic cells, as well as the different events happening withinthe dendritic cells after phagocytosis. Staining for a lysosomal marker (LAMP1) as well

Savina Stoitsova

as MHC Class II (I-Ab) gave the localization patterns of apoptotic cells and MHC Class IImolecules with respect to the endocytic pathway.

References:

Blander, J., Medzhitov, R., Toll-dependent selection of microbial antigens forpresentation by dendritic cells, Nature, February 2006

Lyons, B., Analyzing cell division in vivo and in vitro using flow-cytometricmeasurements of CFSE dye dilution, Journal of Immunological Methods, 2000,147-154

Lyons, B., Cell division tracking using CFSE, a ppt presentation available at HTTP:jcsmr.anu.edu.au/facslab/AFCG/mooloolaba/workshop%20Presentations/CELLDI~1.PPT –

Chemical structure of CFSE available at HTTP: http://www.dojindo.com/newprod/1/cell-v/staining/89gif/cfse.gif