Cellular Immunology 256 (2009) 7985

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Cellular Immunology
journal homepage: www.elsevier.com/locate/ycimm

Assessment of lymphocyte proliferation: CFSE kills dividing cells

and modulates expression of activation markers
J. Latovicka *, V. Budinsky, R. pek, J. Bartunkov
Institute of Immunology, 2nd Medical School and University Hospital Motol, Charles University, V valu 84, 150 06 Praha 5, Czech Republic

a r t i c l e

i n f o

Article history:
Received 4 August 2008
Accepted 22 January 2009
Available online 23 February 2009
Keywords:
3
H-thymidine incorporation
CSFE staining
Expression of activation markers
Immunodeciencies

a b s t r a c t
The measurement of cell proliferation after mitogenic stimulation is an important parameter used in
diagnosis of immunodeciencies in clinical laboratory as well as in various elds of lymphocyte research.
Recent methods try to overcome the radioactive assay using tritiated thymidine (3H) by ow-cytometric
methods using different uorochromes such as CFSE or even to substitute these direct methods by tracing
the expression of cell-membrane activation markers associated with various steps of proliferation cycle.
In our study we compared the 3H assay with CFSE-staining method and expression of activation markers
(CD69, HLA-DR, CD25, CD27, CD71, CD152, CD134 and CD195) on a sample of 128 consecutive patients
and healthy controls evaluated in clinical laboratory. We also tested various concentrations of CFSE and
its impact on proliferation activity and expression of activation markers. We found that CFSE in concentration from 37 nM to 10 lM decreases the proliferative capacity (expressed in cpm 3H assay) due to the
decreased viability of proliferating cells (measured as 7-AAD+) in concentration-dependent manner.
Moreover, CFSE substantially modulates the expression of activation molecules (decreasing CD69, HLADR, CD25 the majority of examinated molecules). We found a good correlation between CFSE-staining
method with 3H assay, if CFSE low population is gated on CD3+ population (correlation coefcient
0.801), but only in samples with stimulation index (SI) higher then 25. In poorly proliferating samples
(SI 6 25) no correlation was found due to several false positive results in CFSE test. Statistically signicant
correlation between proliferation assessed as 3H-thymidine incorporation and expression of activation
markers was found in the case of CD25, CD27, CD38, CD152, CD71, still only in samples with higher
proliferation activity (SI > 25). No correlation was found with CD134, CD195, HLA-DR and CD69. We
conclude that standard assay with 3H-thymidine incorporation is unreplaceable assay in diagnosis
of severe cellular immunodeciencies as CFSE assay have high proportion of false positive results.
Researchers tracing cell-membrane bound molecules on dividing cells stained by CFSE must take into
account that CFSE may substantially modulate the expression of these markers and decrease the viability
of stained cells.
2009 Elsevier Inc. All rights reserved.

1. Introduction
The measurement of cell proliferation after antigenic or mitogenic stimulation is an important parameter used in diagnosis of
various immunodeciencies in routine clinical laboratory as well
as in various elds of lymphocyte research. Measuring incorporation of tritiated thymidine (3H) into lymphocyte DNA is still the
most commonly used approach for determining cell division, despite of disadvantage of dealing with radioactive material. This
method uses the physiological activation of T lymphocytes via T
cell receptor (TCR)/CD3 complex by various polyclonal activators
which initiate a series of intracellular events leading to DNA syn-

* Corresponding author. Fax: +420 2 2443 5962.

E-mail address: jan.lastovicka@lfmotol.cuni.cz (J. Latovicka).
0008-8749/$ - see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.cellimm.2009.01.007

thesis. Phytohemagglutinin (PHA), plant lectin isolated from Phaseolus vulgaris, is the most frequently used mitogen [1].
Novel approaches try to overcome disadvantage of 3H-thymidine uptake assay by the use of cytoplasmic dye carboxyuorescein diacetate succinimidyl ester (CFDA-SE), for the tracking of
lymphocyte division [2].
CFDA SE (also called CFSE) is a non-uorescent dye that passively diffuses into the cytoplasm of cells. Its nonpolar molecule
spontaneously penetrates cell membranes and is converted to anionic CFSE by intracellular esterases, becoming uorescent. The succinimidyl ester group covalently binds to amino groups on
intracellular macromolecules, anchoring the dye. Amine-reactive
coupling of CFSE to proteins results in stable long-term intracellular
retention. CFSE spontaneously and irreversibly couples to cellular
proteins by reaction with lysine side chains and other available
amines. This highly amine-reactive product then forms dye-protein

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J. Latovicka et al. / Cellular Immunology 256 (2009) 7985

adducts that are retained by the cells throughout their development. CFSE is inherited equally by daughter cells after division,
resulting in the sequential halving of mean uorescence with each
generation [3]. When analyzed by ow cytometry, this sequential
halving of uorescence is visualized as distinct peaks and can be
used to track division progression [4]. The mechanism of action
and comparison of CFSE with other dyes that have been used to
track lymphocyte migration and proliferation were described by
Parish [5].
To overcome disadvantages of cellcell culture systems for
assessing lymphocyte proliferation, there are many attempts to
substitute these methods by indirect methods focused on the
detection of cellular markers associated with the proliferation
and activation. Many molecules appear on the surface of lymphocytes after activation and the degree of their expression more or
less correlates with activation status [6], preceding or accompanying cell division. The CD69 antigen is one of the earliest markers
expressed on activated T, B, and natural killer (NK) lymphocytes
following stimulation by a variety of mitogenic agents, reaching
its peak of expression within 24 h [7], but detectable also after
72 h [2].
Besides CD69, stimulation of T cells by mitogen also results in
the upregulation of other surface proteins, such as CD25 (IL-2
receptor), HLA-DR, and CD71 (transferrin receptor) [8].
The close relationship to proliferation was also described for
other surface molecules. The tumor necrosis factor receptor family
includes several important markers of T cell activation including
CD134 (OX40) reaching its peak 72 h after the stimulation [9].
CD152 (CTLA-4) expression on T cells also increases after PHA
stimulation [10].
Finally, receptors CCR5 (CD195) and CCR7 (CD197) can be considered as activation markers.
In this study we investigated the correlation between the surface expression of all these surface antigens and 3H-thymidine
incorporation and CFSE staining on the large group of patients
and healthy donors. Correlations between these methods were
then evaluated.
We found that correlation between CFSE staining and 3H-thymidine incorporation was weak in group of patients with severe
immunodeciencies (stimulatory index (SI) < 25). The best correlation was found between CFSE low gated on CD3+ population. Statistically signicant correlation was found for several activation
markers (CD25, CD27, CD38, CD152, CD71), but only for samples
with SI above 25. Moreover we demonstrate that CFSE is toxic
for dividing cells and CFSE staining substantially affects the expression of activation markers.

CFSE or cultured unstained. These PBMC were used also for FACS
analysis as described in Section 2.5.
2.3. CFSE staining
5-(and-6)-Carboxyuorescein diacetate, succinimidyl ester
(5(6)-CFDA, SE; CFSE) mixed isomers (Vybrant CFDA-SE Cell Tracer Kit) was purchased from Invitrogen (Carlsbad, CA, USA). CFSE
was stored frozen as a 10 mM stock solution until used. A pellet
of 2  106 cells was resuspended in 1 ml of CFSE labeling solution
of concentration ranging from 10 lM to 37 nM and incubated
10 min in 37 C in the dark. After incubation, 1 mL of fetal calf serum was added to each sample followed by 1 min incubation. Cells
were then washed three times with PBS and centrifuged at 400 g
for 10 min at room temperature before resuspension in the culture
medium. PBMC stained with CFSE were analyzed after 72 h of cell
culture on FACS Aria (Becton Dickinson, CA, USA), and data analyzed using FlowJo software. The CFSE low fraction was determined as shown on Fig. 1.
2.4. Lymphocyte proliferation assay
PBMC were cultured in X-VIVO (Cambrex, Belgium), supplemented with 2 mM L-glutamine (Cambrex, Belgium), 500 lM 2mercaptoethanol (Sigma Aldrich, Germany), 10% heat-inactivated
FCS, 1% penicillin/streptomycin (Cambrex, Belgium), in a nal concentration of 1  106 cells/mL. Cells (2  105) suspension were cultured in U-bottom 96-well plates (Nunc, Denmark) with either
5 lg/mL phytohemagglutinin (PHA) (Sigma Aldrich, Germany) or
medium alone. Cultures were incubated at 37 C in a 5% CO2 incubator for 72 h. Eighteen hours before harvesting cells were pulsed
with 2 lCi of 3H-thymidine (Amersham, UK), with uptake being
measured by LKB Rackbeta 1219 (Turku, Finland). Both mean raw
counts per minute (cpm) values of the triplicate as well as the
stimulation index (SI), calculated by dividing the 3H-thymidine uptake values by the corresponding value in control wells, were
calculated.
2.5. Flow cytometry
PBMC were labeled with directly conjugated monoclonal antibodies: CD3 PacicBlue (eBioscience, USA), CD25 Dy747 (Exbio,
Czech republic), CD 27 PE-Dy590 (Exbio, Czech republic), CD38
PC5 (Exbio, Czech republic), CD69 FITC (Exbio, Czech republic)
CD71 FITC (Exbio, Czech republic), HLA-DR PC5 (Exbio, Czech

2. Materials and methods

2.1. Patients
Blood samples from 128 patients of the Institute of Immunology, University hospital Motol in Prague, with various immunological disorders and 40 healthy controls were used in the
experiments. The signed consent from all patients and donors
was obtained and the experiments were performed with an agreement of local ethical committee.
2.2. Lymphocyte separation
Peripheral blood was collected into sodium heparin tubes (8 ml
Vacutainer; Becton Dickinson), diluted with an equal volume of
PBS, then layered over Ficoll-paque (Sigma Aldrich, Germany) density gradient separation solution, and centrifuged at 300g for
30 min at room temperature. The mononuclear cell layer (PBMC)
was removed and washed twice in PBS, counted and labeled with

Fig. 1. Determination of dividing cells based on CFSE low fraction. Comparison of

PHA stimulated PBMC (line) with unstimulated cells (lled).

J. Latovicka et al. / Cellular Immunology 256 (2009) 7985

republic), CD134 APC (eBioscience, USA), CD152 PE (eBioscience,

USA), CD195 PE (eBioscience, USA), CD197 PE (Pharmingen, USA)
for 30 min in the fridge. Samples were analyzed on FACS Aria (Becton Dickinson, CA, USA). From each sample a minimum of 100,000
events was collected and analysed using Flowjo software. Standard
gating procedures using FMO controls were used to identify positive cells.
2.6. Cell viability determination
Cells were stained with 0.25 lg/106 cells of 7-amino actinomycin D (eBioscience, USA), and incubated for 5 min before analysis
on FACS Aria. 7-AAD positive cells were considered as dead.
2.7. Statistical analysis
Paired t-test was employed to assess differences between the
corresponding values in the groups. Spearman correlation was
used to examine the relation between 3H-thymidine incorporation
and ow cytometric analysis (expression of activation molecules
and CFSE staining). p-values of less than 0.05 were considered
signicant.
3. Results
3.1. Cell proliferation and viability
CFSE interacts with various components of cells including cell
membranes, surface glycoproteins, enzymatic systems and DNA.
Its thus conceivable, that it could alter normal cellular functions.
We investigated the inuence of various CFSE concentrations on
proliferation of PHA stimulated lymphocytes. Cells stained with
CFSE showed signicantly impaired proliferation in all tested concentrations, ranging from 37 nM to 10 lM (Fig. 2A). Frequency of
dividing cells (dened as CFSE low fraction) was signicantly lower

81

in samples stained with concentrations of CFSE above 1 lM

(Fig. 2B). This correlated with impaired expression of CD25 and
CD71 (Fig. 3A and B). The viability of cells gradually decreased with
the increasing concentration of CFSE (Fig. 4A and B) in both PBMC
(Fig. 4A) and leukemia cell line L1210 (Fig. 4B).
3.2. Effect of CFSE on the expression of activation markers
As CFSE staining is often used in research laboratories for simultaneous detection of activation markers on dividing cells, we further examined the inuence of various CFSE concentrations on
the expression of activation markers during 4 days of cell culture.
CFSE impaired the expression of CD25 (Fig. 3A) and CD71
(Fig. 3B). On the contrary, the expression of activation marker
HLA-DR and CD27 was increased when higher CFSE concentrations
were used (Fig. 3C and D). Similar increase was also found for
CD134 and CD152 (data not shown). Moreover, we found that CFSE
staining has different effects on cells from healthy donors and patients. An example of this represents the CD3 molecule: while the
CD3 expression by cells from healthy donors was not altered. In patient with aplastic anemia evaluated 3 years after bone marrow
transplantation, CFSE induced the increase of CD3+ cells which
was obvious throughout the whole CFSE concentration range. Increase of CD3 concerned both percent of positive cells and number
of CD3 molecules expressed by one cell (determined as MFI)
(Fig. 5A and B).
3.3. Correlation of 3H-thymidine incorporation, CFSE staining and the
expression of activation molecules
We next evaluated the correlation between lymphocyte proliferation assessed by 3H-thymidine incorporation with proliferation
determined by CFSE staining and with the expression of activation
markers. The CFSE low fraction was determined by comparing
unstimulated control with the PHA stimulated lymphocytes. CFSE

Fig. 2. CFSE labeling signicantly impairs T cell proliferative capacity. (A) Cells from healthy donors were stained with CFSE, cultured for 72 h with PHA and then 3Hthymidine incorporation was measured. Columns marked with asterisk are signicantly different from unstained control (***p < 0.01) (average from three independent
experiments is shown). (B) Flow cytometry histograms of CFSE stained cells.

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J. Latovicka et al. / Cellular Immunology 256 (2009) 7985

Fig. 3. Inuence of various concentrations of CFSE on expression of activation markers. PBMC of healthy donors were stained with various concentrations of CFSE, then
cultured 24, 48, 72 or 96 h in the presence of PHA and then stained for ow cytometry. One representative experiment out of three is shown. (A) Inuence of CFSE staining on
expression of CD25. (B) Inuence of CFSE staining on expression of CD71. (C) Inuence of CFSE staining on expression of HLA-DR. (D) Inuence of CFSE staining on expression
of CD27.

Fig. 4. Inuence of various concentrations of CFSE on cell viability measured by 7-AAD staining. (A) PBMC of healthy donors were stained with various concentrations of CFSE,
then cultivated 24, 48, 72 or 96 h in the presence of PHA and then stained for ow cytometry. Average of three experiments, columns marked with asterisk are signicantly
different from unstained control (**p < 0.05, ***p < 0.01). (B) Tumor cell line L1210 was stained with various concentrations of CFSE and immediately stained for ow
cytometry. Average of three experiments, columns marked with asterisk are signicantly different from unstained control (**p < 0.05, ***p < 0.01).

J. Latovicka et al. / Cellular Immunology 256 (2009) 7985

83

Fig. 5. Effect of CFSE staining on expression of CD3 on PBMC from patient with aplastic anemia. PBMC were either stained with CFSE or not, cultivated 24, 48, 72 and 96 h in
the presence of PHA and then evaluated by ow cytometry. (A) % of CD3 positive cells and (B) mean uorescence intensity.

Fig. 6. Scatterplot of correlation between CPM and CD3+CFSE low fraction. (A) Samples with SI > 25, correlation coefcient 0.801. (B) Samples with SI 6 25, correlation
coefcient 0.467.

Fig. 7. Scatterplot of correlation between CPM and expression of CD25. (A) Samples with SI > 25, correlation coefcient 0.783. (B) Samples with SI 6 25, correlation coefcient
0.313.

was used at the recommended concentration of 1 lM. The best

correlation coefcient (0.807) was observed for analysis of T cells
proliferation by CFSE dilution. The second highest correlation
(0.764) was obtained for CD25 expression. The scatter plots of correlation of CFSE method and CD25 expression with 3H-thymidine
incorporation are shown in Figs. 6 and 7. Correlations between
the MFI values instead of percents of positive cells and 3H-thymidine incorporation yielded similar values. The correlation coef-

cients obtained for all other tested activation markers were

below 0.7 (Table 1).
3.4. Correlation of 3H-thymidine incorporation, CFSE staining and the
expression of activation molecules by immunocompromised patients
To evaluate the feasibility of CFSE staining or detection of activation markers expression in immunocompromised patients who

J. Latovicka et al. / Cellular Immunology 256 (2009) 7985

84

Table 1
Correlation of CPM with CFSE staining and expression of CD markers on PHA
stimulated lymphocytes after 72 h of cell culture. Spearman correlation test was used
for statistical analysis. N represents the number of measured samples. Last two
columns compare the blood samples with SI 6 25 and SI > 25.
All SI

Samples

Samples

SI 6 25
n
CD3+CFSE low
CFSE low
CD25+
MFI CD25
CD3+CD25+
CD27+
MFI CD27
CD3+CD27+
CD38+
CD152+
CD71+
CD134+
CD195+
HLA-DR+
CD69+
*

66
128
100
130
48
116
116
52
34
44
80
44
26
98
22

1
0.807
0.776*
0.764*
0.720*
0.558*
0.604*
0.587*
0.460*
0.577*
0.582*
0.541*
0.236
0.222
0.061
0.03

SI > 25

10
18
18
24

0.467
0.461
0.313
0.189

20
22

0.277
0.492

8
8
8

0.333
0.643
0.262

14

0.305

56
110
82
106
44
96
94
46
26
36
73
36
20
85
16

1
0.801*
0.661*
0.783*
0.795*
0.597*
0.641*
0.610*
0.438*
0.598*
0.551*
0.533*
0.265
0.015
0.032
0.109

Values are statistically signicant (p 6 0.05).

have impaired 3H-thymidine incorporation, we divided all patients

into two groups based on their stimulation index as determined by
3
H-thymidine incorporation. With the exception of CD71 and CD
152, all correlation coefcients in the group of patients with
SI 6 25 were lower than in undivided group and substantially lower than in the group with SI P 25. Several patients with no measurable incorporation of 3H-thymidine (patients with SCID or shortly
after bone marrow transplantation) still showed positive expression of CD69, CD25 and had detectable fraction of CD3+ CFSE low
lymphocytes. This nding indicates that both the CFSE staining
and activation markers expression lack the specicity in diagnosing T cell immunodeciency.
4. Discussion
CFSE has been widely used for tracking the cell division in a
number of cell types both in vitro [11] and in vivo [12]. Detrimental effect of CFSE on lymphocyte function is not generally considered even if it has been already described [13]. Also the toxicity
for tumor cell lines was tested previously [14] by PI staining. The
cell death rate was found to be less than 5% within 16 h of incubation in concentrations 110 lM. On the contrary, our studies
with PBMC revealed substantial impact on cell viability during
72 h of cell culture, reaching up to 50% in concentration range from
5 to 10 lM.
The toxicity and suppression of proliferative responses even in
the concentrations below 1 lM are not considered by the manufacturer. According to the manufacturer recommendation for longterm staining (more than three days) or the use of rapidly dividing
cells requires 510 lM dye and for shorter experiments lower concentration (0.55 lM) is required (Invitrogen, Vybrant CFDA SE
Cell Tracer Kit instructions). The correlation of CFSE staining with
classical test with 3H-thymidine uptake (correlation coefcient
0.829) shows, that the CFSE method does not reach the accuracy
and sensitivity of gold standard 3H-thymidine uptake. Also the
comparison of labor intensity for both methods does not benet
the CFSE staining. Moreover, we show that in severely immunodecient patients, CFSE test may give false positive results.
The detection of the mitogen-induced early activation marker
CD69 has been postulated as a simpler, faster, and safer in vitro
assessment of T-cell function than 3H-thymidine incorporation
[7]. In later study, [15] the level of mitogen-induced T-cell CD69

was compared with the proliferative response, measured by 3Hthymidine incorporation, in a group of healthy controls and a
group of patients known to have T-cell dysfunction. While only
one out of the ve patients exhibited reduced CD69 expression,
all had lower than normal proliferative responses as measured
with 3H-thymidine incorporation. The results indicated that the
CD69 assay lacked specicity in diagnosing immunodeciency.
Da Fulcher et al. studied [2] the correlation of 3H-thymidine uptake
with the expression of some activation markers, including CD69,
which peaks about 24 h after activation. Despite of their ndings,
that CD69 remained highly expressed after 3 days in culture, we
found no correlation with 3H-thymidine uptake by this marker
after 72 h of cell culture. Also another research group found no correlation between the percentage of CD69 expressing lymphocytes
and the amount of 3H-thymidine incorporation [16]. Similarly,
the expression of CD69 did not differ between patients with immunodeciency and control subjects [2] after 3 days in culture. Undivided cells expressed high levels of CD69, which demonstrated that
the designation of CD69 as an early T-cell activation marker is an
over-simplication.
From all tested activation markers CD25 seems to have closer
correlation to CPM, but still not enough for using this method as
an equivalent of 3H-thymidine uptake. Santos and coworkers [17]
examined the feasibility of determining T lymphocyte activation
by measuring IL-2 receptor (CD25) expression by ow cytometry.
They found a good correlation between CD25 molecule expression
and the dose of phytohemagglutinin, however, there was no correlation between CPMs produced by 3H-thymidine uptake and the
percentage of CD25 expressed by cells.
By detecting activation antigens expressed on T cells during the
late-stage differentiation (7296 h) such as HLA-DR or CD71
(transferrin receptor), the percentage of positive lymphocytes
and the amount of 3H-thymidine incorporation were not found
to correlate under any condition tested which is in accordance with
the nding of others [16].
The main advantage of CFSE labeling used in many experimental settings is the possibility of simultaneous tracking the expression of activation molecules on the surface of stained cells both
in vivo and in vitro [2022]. However, according to our results,
CFSE may substantially modulate the expression of many markers.
Some of them like CD25 and CD71 are downregulated and percent
of positive cells decrease during the PHA-induced proliferation and
CFSE labeling. This nding is partially due to the decreased cell viability induced by CFSE, but this is not the only explanation as the
percentage of dead cells is lower than the percentage of appropriate antigen-expressing cells. Surprisingly, other markers as HLADR or CD27 are upregulated in the presence of CFSE. The cause of
this phenomenon is not clear but may be related to the modication of intracellular structures by CFSE which may lead to the
translocation of molecules from cytoplasmic stores to the membrane. Another phenomenon we observed was the fact that cells
from healthy controls and patients may behave differently. The
nice example is the molecule CD3 which was upregulated in cells
from a patient 3 years after bone marrow transplantation for aplastic anemia. The explanation of this particular behavior of CD3 molecule is only hypothetic, but may reect different metabolism or
intracellular trafcing of CD3 molecules within donor cells in the
recipient milieu. This idea is supported by some studies that aberrant lymphocytes have increased expression of intracytoplasmic
CD3 molecules [18]. Taken together, our nding that CSFE may
substantially modulate the expression of membrane molecules
must be taken into account when assessing the membrane bound
molecules on CFSE stained cells, especially in abnormal conditions.
The only method for measurement of lymphocyte proliferation
which was found to be comparable with 3H-thymidine uptake is
probably the bromodeoxyuridine incorporation. The determination

J. Latovicka et al. / Cellular Immunology 256 (2009) 7985

of the percentage of cells in S-phase is dependent upon the detection of a thymidine analog, bromodeoxyuridine (BrdU), which
when added to culture medium is incorporated into DNA during
DNA replication. Motobu [19] found a good correlation between
the 3H-thymidine incorporation assay and BrdU incorporation assay by ow cytometry. Stimulation indexes in proliferative responses to mitogens by the BrdU incorporation assay positively
correlated with those by the 3H-thymidine incorporation assay
(r = 0.899 for PWM and r = 0.934 for Con A). However, BrdU assay
requires intracellular staining and is both more time consuming
and more expensive than 3H-thymidine assay, and therefore less
suitable for routine examination.
Studying cell proliferation by 3H-thymidine incorporation, uorochrome labeling or by indirect investigation of activation-associated cellular markers are methods which cannot be easily
substituted one for another. Since use of CFSE can interfere with
many cellular functions and phenotype, the methods for assessing
cell proliferation and activation have to be carefully chosen with
regard to the purpose of testing.

[7]

[8]

[9]

[10]

[11]
[12]

[13]

[14]

[15]

Acknowledgment
This work was supported by the research project VZ MSM
002162 0812.

[16]

[17]

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