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).
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.
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 unpublished).
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
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
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