CFDAse

In our opinion Carboxyfluorescein diacetate, succinimidyl ester (5(6)-CFDAse) is the best reagent currently available for generation analysis of cells. CFDAse couples irreversibly by the esterases from the cell itself to both intracellular (mainly) and cell-surface proteins by reaction with lysine side-chains and other available amine groups. The daughter cells become half as fluorescent as their parent when this divided. As a result, each division results in the generation of a population of cells that is characterized by half of the cellular fluorescence intensity. These distinct populations of cells can be visualized by the use of flow cytometry (Lyons and Parish, 1994).

In figure.1.A. a typical profile is shown of dividing peripheral blood CD4+ T lymphocytes after 5 days stimulation with CD3/CD28. When different compounds are added to the different cultures the influence on the diving T lymphocytes can be studied. This simply can be done by making overlays of the histograms (figure.1.A.). When a (polyclonal) stimulus is used for cells of a cell line it becomes clear that there is a heterogeneity in the population concerning the resting state of the cells (figure.1.B.).

 

Figure.1.A. CFDAse profiles of peripheral CD4⁺ T lymphocytes stimulated for 5 days with CD3/CD28 in the absence or presence of different compounds (dark profiles). In the histograms of the cells in the presence of the compounds the neutral situation (CD3/CD28) is represented with the open profile. Clear is that the compounds A and D have a negative effect on the division of the T lymphocytes.

Figure.1.B. CFDAse profiles of a CD4⁺ T lymphocyte line that has been stimulated for 5 days with SEB in the presence of monocyte derived dendritic cells. Numbers indicate the number of divisions that the cells underwent.

Sometimes it is difficult to evaluate the results and discriminate the different populations. Or you would like to calculate the initial population that started to divide. To evaluate these results software packages can be used to calculate these numbers for you. There is also literature available that makes clear how you can calculate these numbers yourself.

There are also other dyes, like PKH26 for instance, to detect cell division but CFDAse gives a more homogenous cellular labelling and better resolution of the different generations than the mentioned (membrane marker) PKH26. Sometimes PKH also seems to leak from the cells and 'contaminates' surrounding cells more often than CFDAse. There is no proof that CFDAse is interfering with the reaction to antigens by lymphocytes for instance because of the surface labeling of the antigen presenting cells or lymphocyte by CFDAse. In our hands neither does PKH26 interfere. It is even possible to use both dyes in on experiment. Which makes it possible to discriminate two or three different populations in on experiment (figure.2.). At this moment we are looking into a new dye that would make it possible to perform the evaluation of generations of a third population of cells in one experiment.

Than there is also a weaker fluorescent succinimidyl ester, SNARF-1 carboxylic acid, acetate is similar to that of CFDAse as a cell tracer and indicator of cell division. This ester has a red fluorescence when excited near 488 nm. Its red fluorescence is easily distinguished from the green fluorescence of CFDAse labelled cells. These SNARF dyes have been predominantly used as indicators of intracellular pH.

Figure.2. Initial populations of peripheral CD4+ T lymphocytes were stained with either PKH26 (red population) or CFDAse (green population) and stimulated with CD3/CD28 for 5 days. At day 5 cells are evaluated by flow cytometer.

  A.                                                             B.

Figure.3. Cytokine production profile of two distinct populations of lymphocytes. A. IL-4/IFN-y profile of CD4⁺CD45RO⁺ lymphocytes that have the lowest fluorescence intensity for CFDAse (five subsequent divisions, indicated with 5 in figure CFDA.1.) and have been restimulated with PMA/ionomycin for 6 hours in the presence of Brefeldin A. B. IL-4/IFN-y profile of CD4⁺CD45RO⁺ lymphocytes that have divided two times (indicated with 2 in figure CFDA.1.) and have been restimulated with PMA/ionomycin for 6 hours in the presence of Brefeldin A.

Using CFDAse and flow cytometric analysis it is possible to detect eight to ten generations of lymphocytes. Also in combination with the phenotype of the cells and other markers like cytokines in a certain generation of the dividing population (figure.3.)(Richter et al. 1999, Schuitemaker et al. unpublished).
The CFDAse is also used in homing experiments by transplanting labelled cells into animals. And even the staining of plant cells and bacteria is an option. Because it is the first division that results in the largest change in fluorescence intensity, this method is particularly useful for detection of a subset of cells within the population that is hardly dividing or has a very low frequency, like antigen specific lymphocytes.

Of course there are many other ways to detect cell proliferation. As there are the incorporation of radioactive ³H-thymidine or the use of 5-bromo-2'-deoxyuridine (BrdU, a thymidine analogue). Both methods don't show the different generations of cells though. The thymidine incorporation doesn't allow the evaluation of phenotype or other cell markers. The BrdU only facilitates the identification of cells that have progressed through the S-phase of the cell cycle during the BrdU labelling period. And needs a secondary reagent to show the incorporated BrdU.
 

Although we did our best we can not be held responsible for any mistakes or typing errors.
 Suggestions, literature and protocols are welcome at icstaining@schuitemaker.info. Copyright 2000 - 2005.
Page was last updated: 05-03-2005