Use of the Intracellular Fluorescent Dye UNIT 4.

9
CFSE to Monitor Lymphocyte Migration
and Proliferation
Christopher R. Parish,1 Megan H. Glidden,1 Ben J. C. Quah,1 and Hilary S.
Warren1
1
Australian National University, Canberra, Australia

ABSTRACT
The stable incorporation of the intracellular fluorescent dye 5-(and -6)-carboxyfluorescein
diacetate succinimidyl ester (CFSE) into cells provides a powerful tool to monitor cell
migration, and to quantify cell division, because of the sequential decrease in fluorescent
labeling in daughter cells. CFSE-labeled lymphocytes have been used to analyze the
relationship between cell division and differentiation of cell function, and cell prolifera-
tion versus apoptosis, both in vivo and in vitro, and have allowed analysis of the site of
response to antigens in vivo. Curr. Protoc. Immunol. 84:4.9.1-4.9.13.  C 2009 by John

Wiley & Sons, Inc.

Keywords: CFSE r cell division r cell tracking r lymphocyte migration r
lymphocyte positioning

INTRODUCTION
The stable incorporation of the intracellular fluorescent dye 5-(and -6)-carboxyfluorescein
diacetate succinimidyl ester (CFSE) into lymphocytes (Basic Protocol) is a powerful tool
to monitor lymphocyte migration in vivo, and to quantitatively analyze cell division both
in vivo and in vitro (Support Protocol). The stability of CFSE labeling allows monitoring
of lymphocytes over a period of months in vivo. Cell division results in sequential halving
of fluorescence, and up to 8 divisions can be monitored before the fluorescence is de-
creased to the background fluorescence of unstained cells. The relationship between cell
division and cell function is readily measured at the time of analysis by using a cell func-
tion marker (cell surface or intracellular protein) labeled with an alternate fluorochrome.
Similarly, T and B lymphocyte subsets and NK cells can be individually analyzed for
cell division in a complex population by using appropriate cell surface markers. CFSE
remains associated with apoptotic cells for several days, and these can be analyzed to-
gether with live cells by appropriate electronic gating (by size and granularity) on the
flow cytometer. Since halving of fluorescence occurs in daughter cells, by calculating
the proportion of cells in each division peak and dividing by the expected progeny at
those divisions (2, 4, 8, 16, etc.), the number of cells that have entered division can be
calculated. This gives a precursor frequency estimate of responding cells in the cultures.

CFSE LABELING OF LYMPHOCYTES BASIC

PROTOCOL
This protocol describes methods for labeling high or low numbers of lymphocytes with
CFSE. Steps are provided to use CFSE-labeled cells in cell transfer studies in vivo or
as cells to be cultured in vitro. If analysis of cell migration is a goal of the experiment,
specific guidelines for positioning of CFSE-labeled lymphocytes in lymphoid organs
or other tissues are provided in this protocol. A Support Protocol for flow cytometric
analysis of CFSE-labeled cells follows. In Vivo Assays
for Lymphocyte
Function

Current Protocols in Immunology 4.9.1-4.9.13, February 2009 4.9.1

Published online February 2009 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/0471142735.im0409s84 Supplement 84
Copyright C 2009 John Wiley & Sons, Inc.
 Materials
Experimental animals or human peripheral blood or cultured lymphocytes
Phosphate-buffered saline (PBS; APPENDIX 2A), pH 7.4
Hanks’ balanced salt solution (HBSS), pH 7.4 (APPENDIX 2A)
PBS (APPENDIX 2A) containing 5% (v/v) heat-inactivated FBS
5 mM CFSE stock solution (see recipe)
Antigens and mitogens of interest
0.5 mM disodium EDTA in PBS (APPENDIX 2A)
1-ml syringes
25-G needles
Fluorescence microscope with filters for fluorescein
Razor blade
Additional reagents and equipment for removal of mouse lymphoid organs
(UNIT 1.9), preparation of mononuclear cell suspensions (UNIT 3.1), and isolation of
peripheral blood mononuclear cells (UNIT 7.1), immunohistochemistry (UNIT 21.4),
culturing mouse (UNITS 3.10 & 3.12), or human (UNITS 7.10 & 7.11) lymphocytes, and
counting cells using a hemacytometer (APPENDIX 3A)
Label lymphocytes with CFSE
1a. For high cell numbers: Prepare lymphocytes using the techniques described in UNIT 1.9
(for mice; removal of lymphoid organs), UNIT 3.1 (preparation of cell suspensions),
and UNIT 7.1 (preparation of PBMC), at a concentration of 50 × 106 cells/ml in
either PBS (without serum) for human PBMC or HBSS (without serum) for mouse
lymphocytes.
1b. For low cell numbers: Resuspend freshly isolated lymphocytes in PBS containing
5% FBS at concentrations from 0.5 × 106 cells/ml to 10 × 106 cells/ml.
At low cell concentrations it is absolutely essential that there be protein present to buffer
the toxic effect of CFSE.
Cultured lymphocytes that are quiescent at the end of primary culture are labeled directly
in their culture medium (containing 10% FBS) after equilibrating to room temperature.
2. Dilute the stock 5 mM CFSE solution 1/100 in PBS (to give a 50 μM solution). Add
110 μl of this solution per milliliter of cells (to give a final concentration of 5 μM),
and mix rapidly. After 5 min at room temperature add 10 vol of PBS containing 5%
FBS, centrifuge cells 5 min at 300 × g, 20◦ C, and remove the supernatant. Wash three
times, each time by resuspending in 10 vol PBS containing 5% FBS, centrifuging
5 min at 300 × g, 20◦ C, and removing the supernatant.
Labeling with CFSE occurs rapidly, and it is essential that CFSE be dispersed as evenly
and quickly as possible so that cells are uniformly labeled. One strategy to achieve this
is to add the cell suspension into the bottom of a 10-ml plastic tube, then, while holding
the tube almost horizontally, add the CFSE solution to a nonwetted portion of the plastic
at the top of the tube. The tube is then capped while 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 10 μM and add an equal volume to the
cell suspension while vortexing. If this strategy is used for high cell numbers, prepare the
lymphocytes at 100 × 106 cells/ml instead of 50 × 106 cells/ml.
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 labeling
Use of CFSE to cultured lymphocytes with CFSE, the cells are washed in PBS and then incubated for
Monitor
Lymphocyte 5 min in 0.5 mM EDTA in PBS to dissociate any aggregates, and washed once more in
Migration and PBS before resuspending in culture medium for restimulation in culture.
Proliferation

4.9.2
Supplement 84 Current Protocols in Immunology
 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 24 hr after labeling.
Perform in vivo transfer of CFSE-labeled lymphocytes
3. Resuspend CFSE-labeled lymphocytes in tissue culture medium lacking added pro-
tein (no serum), and, using a 1-ml syringe equipped with a 25-G needle, inject
intravenously (i.v.) into recipient animals. In the case of mice, inject into the lateral
tail vein, with 1 × 106 to 40 × 106 cells being injected into each recipient mouse in
a volume of 0.1 to 0.2 ml.
There is a linear increase in the number of CFSE-labeled cells entering mouse lymphoid
organs with the transfer of up to 50 × 106 cells, but when greater numbers of cells are
transferred the system appears to become saturated.
If extensive proliferation of the CFSE-labeled population is anticipated, it is essential
that the cells have an independent marker for detection from host cells that does not
decrease with cell division. This is most commonly satisfied by using cells that have a
CD45 allotypic difference from host animals, e.g., injection of CFSE-labeled CD45.1+
cells into CD45.2+ host animals (see Critical Parameters and Fig. 4.9.1).
If, on the other hand, in vivo migration of lymphocytes is being investigated under condi-
tions of minimal cell division, it is possible to independently track two different lymphocyte
populations in the same animal by labeling the cells to different fluorescence intensities
with CFSE (Lyons, 1999). One population is labeled with 5 μM CFSE (see step 1a or 1b
above) and the other population with one-quarter (1.25 μM) or one-sixteenth (0.312 μM)
the normal CFSE-labeling concentration.
If one plans to examine the CFSE-labeled cells <24 hr after they have been transferred
into recipient animals, in order to avoid off-scale fluorescence intensities on the flow
cytometer, the lymphocytes should be labeled with one-quarter (1.25 μM) or one-eighth
(0.625 μM) the normal CFSE-labeling concentration (see step 1 or 2 above).

4. Detect the positioning of CFSE-labeled lymphocytes within lymphoid organs and

other tissues by fluorescence microscopy with filter settings for fluorescein, or
by immunohistochemistry (UNIT 21.4) using fluorescein-specific antibodies (Garton
and Schoenwolf, 1996; Graziano et al., 1998). For fluorescence microscopy de-
tection of CFSE-labeled cells, remove organs from animals, cut ∼3-mm sections
of the organs with a razor blade, and place the sections on microscope slides for
examination.
CFSE fluorescence is rapidly quenched when tissue sections are viewed by fluorescence
microscopy and is totally quenched when the sections are treated with conventional his-
tological stains. For short-term positioning studies of relatively low resolution, the DNA-
intercalating dye H33342 is recommended as a highly fluorescent, quenching-resistant
dye for labeling lymphocytes (Parish, 1999).
If high-resolution positioning studies are required, immunohistochemical detection of
CFSE-labeled cells in tissue sections is recommended.

Culture lymphocytes
5. Resuspend CFSE-labeled lymphocytes in culture medium and stimulate in vitro with
antigens or mitogens of interest.
Procedures for culturing mouse and human lymphocytes are detailed in UNITS 3.10 & 3.12,
and UNITS 7.10 & 7.11, respectively.

Harvest cells
6a. For in vivo harvesting: Collect lymphoid organs, and make single-cell suspensions
(UNITS 1.9 & 3.1). In Vivo Assays
for Lymphocyte
Function

4.9.3
Current Protocols in Immunology Supplement 84
 A
1000 104
12000 lymphocytes 1200 CD8α + cells
800

CD8α-APC-A
103

# Cells
9000 900
SSC-A

600

# Cells
6000 102 600
400
1
200 3000 10 300

0 0 100 0 0
0 200 400 600 800 1000 100 101 102 103 104 10 101 102 103 104 100 101 102 103 104
FSC-A CFSE-A B220-PerCP-Cy5-5-A CFSE-A
104 12000 104
Hoechst 33258-A

live cells Vα-2+ cells

3
100
3 10
10 9000

Vα-2-PE-A
80
# Cells

# Cells
102 6000 10 2 60
92.5% 12.2% 40
101 3000 101
20

10 0 0 100 0
0 200 400 600 800 1000 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
FSC-W CFSE-A CFSE-A CFSE-A
CD45.1-PE-Cy7-A 104 40 CD45.1+
3
cells
10 30

# Cells
102 20
31.4%
101 10

100 0 0
10 101 102 103 104 100 101 102 103 104
CFSE-A CFSE-A
B
CFSE-OT-I CD8+ cells CFSE-OT-I CD8+ cells Unlabeled-CD8+ cells
+ OVA + PBS + 20 μg OVA

2 μg 20 μg 200 μg
100 100 100
80 80 80
% of Max

60 60 60
40 40 40
20 20 20
0 0 0
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
CFSE-A

Figure 4.9.1 Progressive gating strategy for detection of maximum T cell divisions in vivo. OVA-specific Vα2+ , CD8α+
T cells were purified from OT-I mice (bred on a CD45.1 background) and labeled with CFSE. T cells (5 × 106 ) were
then intravenously injected into C57BL/6J mice (which have a CD45.2 background). At a time point 2 hr later, mice were
intravenously injected with either 2, 20, or 200 μg of OVA (in PBS) or PBS alone as a control. After 3 days, splenocytes
were harvested, labeled with Hoechst 33258 (at 1 μg/ml for dead cell discrimination) and APC-conjugated anti-CD8α,
PerCP-Cy5.5-conjugated anti-B220, PE-conjugated anti-Vα2, and PE-Cy7 conjugated anti-CD45.1 monoclonal antibodies,
and 1 × 106 cells were collected on an LSR-II (BD biosciences) for analysis. (A) Progressive gating on subpopulations
of splenocytes reveals higher resolution of CFSE profiles of transferred cells (with CD8α+ , CD45.1+ viable cells giving
the highest resolution). (B) Detection of division of transferred CFSE-labeled OT-I CD8+ T cells to increasing amounts of
antigen (OVA).

4.9.4
Supplement 84 Current Protocols in Immunology
6b. For in vitro harvesting: Harvest cells from culture, wash once in 3 ml PBS, resuspend
in 2 ml 0.5 mM EDTA/PBS, and incubate for 5 min at 37◦ C to dissociate aggregates.
Centrifuge cells 5 min at 30 × g, 20◦ C, resuspend in PBS containing 5% FBS,
and transfer to tubes suitable for use with the flow cytometer. If the cells are to be
counted manually with a hemacytometer (APPENDIX 3A), retain a small sample on ice
in a separate tube or V-well plate.
Cells for analysis on the flow cytometer are not fixed with paraformaldehyde, but are kept
on ice until analyzed.
For multiple cultures, it is convenient to use 96-well V-well plates. For cultures harvested
from 96-well flat-well plates, an aliquot of supernatant from each of the wells is first
discarded so that the triplicate cultures can be combined into 1 well of the 96-V-well
plate. Subsequent washing and treatment with EDTA then occurs in 150-μl volumes in
these wells.
The plates are centrifuged 2 min at 300 × g, 4◦ C in a plate holder. The supernatant is
removed using a microtiter pipettor by following the meniscus down while aspirating until
the tip reaches the intersection of the vertical wall and the top of the V-base of the well.
The pellet is mixed by agitating the plate over a vortex mixer prior to adding the next wash
solution with a multipipettor.

ANALYSIS OF CFSE-LABELED CELLS BY FLOW CYTOMETRY SUPPORT

PROTOCOL
This protocol summarizes the steps used to analyze CFSE-labeled cells in order to
quantify cell division. The proportion of cells in the individual CFSE peaks can be
determined manually or by applying software that deconvolutes the peaks. Guidelines
for either approach are provided in this protocol. The Critical Parameters section of
this unit describes important parameters related to the analysis of CFSE-labeled cells
that have been transferred in vivo or cultured in vitro following labeling. Additional
information about flow cytometric analysis of cells can be found in Chapter 5. In this
protocol, the choice of which other labeling procedures are to be used (e.g., cell surface
staining, intracellular staining for cytokines, identification of apoptotic cells) depends on
the goals of the experiment at hand.

Materials
CFSE-labeled cells (see Basic Protocol)
Analytical flow cytometer capable of 3-color fluorescence
Cell sorting flow cytometer (for some applications)
Additional reagents and equipment for cell surface staining of lymphocytes
(UNIT5.3), intracellular cytokine staining (UNIT 6.24), analytical flow cytometry
(UNIT 5.4), flow cytometric analysis of apoptosis (UNIT 3.17), and cell sorting flow
cytometry (UNIT 5.1)
1. Perform flow cytometric staining steps suited to the experiment being performed
(e.g., cell surface staining, UNIT 5.3; intracellular cytokine staining, UNIT 6.24; Hoechst
33258 staining or propidium iodide staining for measurement of apoptosis, UNIT 3.17).
Perform flow cytometric analysis (UNIT 5.4).
CFSE is a fluorescein-based dye. Labeling lymphocytes for cell surface staining or in-
tracellularly for cytokines requires the use of an alternative fluorochrome such as PE,
PE-Cy7, PerCp-Cy5.5, APC, APC-AlexaFluor 750 etc. (see Table 6.21.2).

For software-based calculations

2a. Deconvolute CFSE peaks using appropriate software.
Several software programs, such as Profit for Macintosh (Quantumsoft), Peakfit for PC In Vivo Assays
(SPSS Sciences), and ModFit (Verity Software House; see Internet Resources) are avail- for Lymphocyte
able for deconvoluting CFSE peaks. Programs such as Flowjo (Tree Star; see Internet Function

4.9.5
Current Protocols in Immunology Supplement 84
 Resources) can also be used on both a Macintosh and PC for analyzing CFSE profiles. For
more detailed instructions on analysis of CFSE profiles using software refer to Hawkins
et al. (2007).

For manual calculations

2b. Record the geometric mean fluorescence of the control CFSE-stained cells and the
unstained cells of both the control and stimulated cultures.
For example, one might have a geometric mean fluorescence of 600 for CFSE staining
of control unstimulated cells compared to an autofluorescence control of 2 for stimulated
unstained cells.
Note that the autofluorescence value of the unstained cells is greater for the dividing
compared to the control nondividing cells.

3b. Subtract the geometric mean fluorescence of the appropriate control from that of the
CFSE control cells (e.g., 600 − 2 = 598).
4b. Convert this value to its base 10 logarithm (in the above case, 2.776).
5b. Determine the geometric mean fluorescence of daughter populations. As these occur
at 1/2, 1/4, etc. of the undivided peak, subtract 0.3 log10 units.
Thus, continuing with the above example, the successive peaks are at 2.476 (division 1),
2.176 (division 2), 1.876 (division 3), 1.576 (division 4), 1.276 (division 5), 0.976 (division
6), and 0.676 (division 7).

6b. Determine the boundaries of the peaks.

The boundaries are midway between successive peaks and are therefore 0.15 log10 units
each side of the peak. Therefore, the lower (left) boundary for each peak is as follows:
2.626 (undivided control cells), 2.326 (division 1), 2.026 (division 2), 1.726 (division 3),
1.426 (division 4), 1.126 (division 5), 0.826 (division 6), and 0.526 (division 7). Next, the
antilog of these figures is determined: 423, 212, 106, 53.2, 26.7, 13.4, 6.7, and 3.4. To
these values are added the geometric mean autofluorescence (2 in this example) to give
425, 214, 108, 55.2, 28.7, 15.4, 8.7, and 5.4.
7b. Apply these markers using a flow cytometry program (such as Cell Quest, Becton
Dickinson) and determine the % of cells in each peak.
8b. Determine the actual number of cells in each peak from the manual counts or counting
bead analysis.
9b. Divide the cell numbers in each peak by the expected progeny for those divisions.
Thus, divide by 2 for 1 division, divide by 4 for 2 divisions, divide by 8 for 3 divisions,
etc.
The total of these numbers gives the number of progenitor cells and can be compared with
the number of cells in control cultures.
In some cases applying the above calculations gives markers that are not aligned exactly
over the peaks. When the calculations are redone using division 1 as a reference peak,
the markers then align correctly. It appears that during the transition from undivided to
the first division there is some loss of incorporated CFSE. This appears to occur with
some cell types more than others. The example given in Figure 4.9.2 for human peripheral
blood lymphocytes responding to phytohemagglutinin is a case in point. You will note that
the geometric mean for the undivided cells is 763, which is more than twice that of the
peak in division 1. The remainder of the peaks are close to the expected halving with each
division.
Use of CFSE to This quantitative analysis of cell division giving the number of progenitor cells provides
Monitor
Lymphocyte useful information. First, the lymphocyte population may be stimulated to divide and
Migration and to undergo activation-induced cell death. Dead cells retain the CFSE dye, and thus the
Proliferation division number in which death of a cell occurred can be readily determined. Dead cells

4.9.6
Supplement 84 Current Protocols in Immunology
 M8
M7
M6
M5
M4
M3
M2
M1

0
100 101 102 103 104

Division % at
Marker Left Right Geometric number Progeny each
(M) boundary boundary mean % (i) 2i Progenitors division
1 437 9646 763.13 18.10 0 1 18.10 70.73
2 220 437 310.07 4.24 1 2 2.12 8.28
3 111 220 153.92 5.66 2 4 1.415 5.53
4 58.29 111 77.57 11.80 3 8 1.475 5.76
5 29.43 58.29 40.89 23.47 4 16 1.467 5.73
6 16.55 29.43 22.44 26.22 5 32 0.819 3.20
7 9.31 16.55 13.53 12.05 6 64 0.188 0.73
8 1 9.31 7.63 0.74 7 128 0.006 0.02

Figure 4.9.2 CFSE profiles for PBMC labeled at 1 × 106 /ml in PBS containing 5% FBS with 5 μm
CFSE for 5 min and cultured with 5 μM phytohemagglutinin for 4 days. The unfilled peak on the
right at 7.6 × 106 fluorescence units corresponds to the control unstimulated CFSE-labeled cells.
The unfilled peak on the left at 2 × 100 fluorescence units corresponds to the autofluorescence of
unlabeled cells. The calculated marker positions are shown: M1 (undivided), M2 (1 division), M3
(2 divisions), M4 (3 divisions), M5 (4 divisions), M6 (5 divisions), and M7 (6 divisions).

can be identified by forward and side scatter or by propidium iodide uptake. Comparison
of the number of progenitor cells in the stimulated and control cultures will show if
significant cell death has occurred. Second, the percentage of progenitor cells in divisions
1 through 7 compared to divisions 0 through 7 is an estimate of the precursor frequency,
i.e., the proportion of the lymphocyte population with reactivity to a particular antigen or
mitogen.
In the experiment shown in Figure 4.9.2, 29.63% of cells have entered division. The
limitation using this technique for the determination of precursor frequencies is the number
of divisions that occur before the fluorescence of the dividing cells merges with the
autofluorescence of unstimulated cells.

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2A; for suppliers, see APPENDIX 5.

CFSE solution
Dissolve 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE; Molec-
ular Probes) in dimethyl sulfoxide (DMSO) at a final concentration of 5 mM. Store
in suitably sized aliquots up to at least 12 months at −20◦ C.
The stock solution can be refrozen provided this is done as soon as possible after thawing.
In Vivo Assays
Prepare the substock of diluted CFSE just before use. Do not refreeze the substock. for Lymphocyte
Function

4.9.7
Current Protocols in Immunology Supplement 84
 COMMENTARY
Background Information
Unlike most other cells in vertebrates, the
cells of the immune system have the remark-
able ability to continually migrate throughout
the body and position themselves in specific
locations within tissues, particularly within
lymphoid organs. In addition, following con-
tact with antigen, there is a rapid clonal expan-
sion of antigen-specific T and B lymphocytes,
with the migration and positioning behavior of
the proliferating lymphocytes often being very
different from their precursors.
In order to fully understand a functioning
immune system, techniques are required that
can simultaneously follow lymphocyte prolif-
eration, lymphocyte migration into different
lymphoid organs, and the positioning pattern
of the lymphocytes within lymphoid organs.
Early studies employed radioactive markers
to measure lymphocyte proliferation and fol-
low lymphocyte migration. [3 H]thymidine has
been widely used to follow lymphocyte pro-
liferation, both in vitro (UNIT 7.10) and in vivo.
However, this approach suffers from the disad-
vantage that it is difficult to assess the subpop-
ulation of lymphocytes that are proliferating,
tedious autoradiography procedures being re-
quired to enumerate the proliferating cells.
Furthermore, the procedure only measures
those cells that are in S phase at the time of
[3 H]thymidine addition. Bromodeoxyuridine
(UNIT 4.7) is an excellent reagent for measur-
ing lymphocyte turnover in vivo, but yields
limited migration and positioning information.
The γ -emitting isotope 51 Cr has been used for
many years to follow the distribution pattern
of injected lymphocytes in vivo but has a num-
ber of technical limitations (Ford, 1978), and
yields no information about the phenotype,
proliferation status, and positioning pattern of
the injected cells.
During the last 25 years, at least 14 differ-
ent fluorescent dyes have been used to moni-
tor lymphocyte migration (Parish, 1999), with
the fluorescein-based dye CFSE emerging as
the most versatile fluorescent dye. CFSE is a
membrane-permeant dye that can very stably
label cells by covalently coupling to intracellu-
lar molecules. CFSE was originally developed
as a long-term tracking dye for lymphocyte
migration studies (Weston and Parish, 1990).
It soon became apparent that a major advan-
Use of CFSE to tage of CFSE is that it can be used to monitor
Monitor lymphocyte proliferation, due to the progres-
Lymphocyte
Migration and sive halving of CFSE fluorescence in cells fol-
Proliferation lowing cell division (Lyons and Parish, 1994).
4.9.8
Supplement 84
Thus, CFSE is an extremely valuable reagent
for immunological studies, as lymphocytes la-
beled with CFSE can be simultaneously mon-
itored for their proliferation status (either in
vitro or in vivo) and their migration and posi-
tioning behavior in vivo.
There is some confusion in the literature
regarding the nomenclature for CFSE. The
compound used to label cells is carboxyflu-
orescein diacetate succinimidyl ester, and thus
a more appropriate acronym would be CFDA-
SE rather than CFSE. Unfortunately, when the
authors first obtained the dye from Molecular
Probes in the late 1980s, it was listed in the
catalog as CFSE and this acronym was used
in the first publications from the authors’ lab-
oratory reporting the use of the dye. In more
recent Molecular Probes catalogs, the dye is
listed as CFDA-SE, but in order to be con-
sistent and avoid confusion, the authors have
continued to use the CFSE acronym in all of
their publications.
To fully appreciate the use of CFSE as
a dye for studying lymphocyte proliferation
and migration, it is important to understand
the molecular basis of cell labeling by CFSE.
Figure 4.9.3 depicts the various stages in the la-
beling of cells with CFSE. Carboxyfluorescein
diacetate succinimidyl ester (CFDA-SE, see
Fig. 4.9.3), because of two acetate side chains,
is nonfluorescent and highly membrane per-
meant. Thus, this compound is rapidly taken
up by cells, although due to its lipophilic nature
CFDA-SE also freely exits from cells. Once
inside cells, intracellular esterases remove the
acetate groups. The resultant carboxyfluores-
cein succinimidyl ester (CFSE, see Fig. 4.9.3)
is highly fluorescent and, due to its reduced
lipophilicity, it exits from cells at a much
slower rate. This slower exit rate also pro-
longs the time available for CFSE to covalently
couple to intracellular molecules. The succin-
imidyl side chain of CFSE is highly reactive
with amino groups, resulting in carboxyfluo-
rescein (CF) being coupled, via a very stable
amide bond, to the amino groups of intracel-
lular molecules. In some cases, CF is coupled
to molecules to form conjugates (CFR1) that
are rapidly degraded or still exit through the
plasma membrane, whereas in other cases the
CF conjugates (CFR2) are stable and unable
to exit from the cell (see Fig. 4.9.3). It is these
long-lived conjugates that allow stable label-
ing of cells with CFSE to be achieved.
Since the mid-1990s many laboratories
have used CFSE to track lymphocyte cell
Current Protocols in Immunology
 O CFDASE O
CH C O O O C CH
3 3
O
O 6
O cell exterior
NO C5
O O
cell membrane

HO O OH

O O R1 NH O CFR1
CH3 C O O O C CH3 HO O OH O
6
O R1 NH C 5
O
O O 6 O O
O 6 esterases
NOC 5 NOC5
O O O O HO O OH
CFDASE CFSE R2 NH
O CFR2
O
cytosol 6
R2 NH C 5
O

Figure 4.9.3 A schematic representation of the various stages in the labeling of cells with
carboxyfluorescein diacetate succinimidyl ester. For details see text. The size of the arrows in the
figure is proportional to the rate of diffusion of the different molecules through the cell membrane.
The carbonyl group located between positions 5 and 6 on the benzene ring is to indicate that the
dye is a mixture of 5- and 6-carbonyl structural isomers. Figure reproduced from Parish (1999)
with kind permission of the publishers (Blackwell Science Asia).

division both in vitro and in vivo (reviewed
in Hasbold et al., 1999; Parish, 1999; Warren,
1999; Lyons, 2000; Quah et al., 2007). An im-
portant feature of these studies is that many
biological processes in lymphocytes, such as
T cell cytokine production, B cell Ig isotype
switching, T cell apoptosis, and cell surface
molecule expression have been shown to be
very division-dependent. A particularly pow-
erful application of CFSE-labeling has been
to monitor T lymphocyte proliferation in re-
sponse to a range of co-stimulatory signals
(Gett and Hodgkin, 2000). Based on their ex-
perimental data, Gett and Hodgkin derived an
elegant mathematical model for the assess-
ment of the effect of different signals on T cell
clonal expansion. The three key variables that
emerged as determining the magnitude of the T
cell proliferative response were the time to first
division, the subsequent rate of division, and
the rate of apoptosis, with different cytokines
and co-stimulatory molecules having unique
effects on these three parameters. Although the
majority of studies have used CFSE to follow
lymphocyte division, the proliferation of cell
types as diverse as fibroblasts, airway smooth
muscle cells, and bacteria has been measured
using CFSE (Khil et al., 1997; Ueckert et al.,
1997; Sukkar et al., 2004). CFSE labeling has
also been reported for tumor cell tracking (von
Horsten et al., 2000), lysis of leukemic precur-
sor cells (Jedema et al., 2004), nuclei label-
ing (Hasbold and Hodgkin, 2000), hematopoi-
etic stem cell proliferation (Oostendorp et al.,
2000), and the in vitro and in vivo assessment
of cytotoxicity (Marzo et al., 2000; Hermans
et al., 2004; Stambas et al., 2007).
Critical Parameters
CFSE is toxic to cells at high concentra-
tions, and it is therefore essential to deter-
mine the optimum labeling conditions that
give good fluorescence and preserve normal
function (Quah et al., 2007). For human pe-
ripheral blood lymphocytes at concentrations
below 10 × 106 /ml, reliable labeling is best
achieved with the cells resuspended in PBS
containing 5% FBS and using CFSE at a final
concentration of 5 μM. These conditions give
a fluorescence intensity between the second
and third decade when measured at 4 days.
Labeling exceeding the third decade gives im-
paired functional responses. This is illustrated
in Figure 4.9.4, showing the relationship, af- In Vivo Assays
ter 4 days culture, between the amount of for Lymphocyte
CFSE in control unstimulated human PBMC Function

4.9.9
Current Protocols in Immunology Supplement 84
 50

40

% of cells in division
30

20

10

0
0 500 1000 1500 2000 2500
Geometric mean fluorescence
of undivided cells

Figure 4.9.4 The relationship between the geometric mean fluorescence of CFSE in undivided
human PBMC and the response of CFSE-labeled PBMC to 5 μM phytohemagglutinin, measured
as the percentage of cells that have entered division after 4 days culture. Data were calculated as
described in Figure 4.9.2.

and the response of the CFSE-labeled cells to
stimulation with 5 μM phytohemagglutinin.
These data are a compilation of that obtained
in an experiment comparing labeling at differ-
ent CFSE concentrations (1 μM to 7.5 μM)
and lymphocyte concentrations (0.5 × 106 to
50 × 106 /ml), and in the presence or ab-
sence of added protein. For any given CFSE
concentration, the presence of protein reduces
the amount of CFSE incorporated. Labeling
is proportional to CFSE concentration and is
essentially complete by 5 min.
Uniform labeling with CFSE is essential
to obtain clearly defined peaks following di-
vision. That is, the coefficient of variation
(CV) of the CFSE-labeled control unstimu-
lated lymphocytes must be small. This is in
part determined by the uniformity in cell vol-
ume of the population, and in part by the tech-
nique for mixing CFSE with the cells. For
some applications it is useful to sort the cells
for uniform staining following CFSE-labeling
(Nordon et al., 1999) prior to culture. Cul-
tured lymphocytes can also be labeled with
Use of CFSE to CFSE, but it is essential to label them when
Monitor
Lymphocyte they have reverted to small cells and are qui-
Migration and escent. CFSE-labeling of cultured human NK
Proliferation cells has allowed an analysis of cell surface
4.9.10
Supplement 84
receptors that stimulate division in secondary
culture (Warren, 1999; Warren and Kinnear,
1999).
Flow cytometry
Analysis of in vivo–transferred cells. When
examining CFSE-labeled lymphocytes in cell
suspensions from in vivo experiments, ideally
0.5 to 1 × 106 events are collected to ensure
sufficient numbers of transferred cells are col-
lected for analysis (these can be ∼1% of the
total population). Care should also be taken
not to gate out the highly fluorescent CFSE-
labeled cells during data acquisition. An alter-
native approach, which reduces data-storage
requirements, is to initially collect 25,000
events in order to calculate the percentage of
donor CFSE+ cells in the sample. One should
then collect 5000 CFSE+ events separately, us-
ing electronic gating, to allow an assessment
of the division status and phenotype of CFSE-
labeled cells.
In this way, CFSE can be used to easily
identify the transferred cells, provided they
have not undergone extensive division. If,
on the other hand, CFSE is used to deter-
mine the proliferative response of the trans-
ferred cells, an independent marker, such as a
Current Protocols in Immunology
CD45 allotype, should also be employed to al-
low definitive identification of the adoptively
transferred cells from host cells, since CFSE
fluorescence may be similar to background
auto-fluorescence in extensively divided cells.
Figure 4.9.1 provides an example of how pro-
gressive gating strategies can be used to dis-
criminate extensively divided CFSE-labeled
cells from host cells. With in vivo experiments,
the number of events that need to be collected
for analysis depends on four key factors: the
number of CFSE-labeled cells that have been
injected, the time since cell transfer, the organ
being examined, and the extent to which the
cells have divided. The latter factor is mainly
important if assessing cell division (not mi-
gration) is of prime importance, as each divi-
sion will result in a cell population of different
fluorescent intensity. Thus, to adequately re-
solve each fluorescent peak, more events will
need to be collected as more divisions (within
the detectable range) occur. Usually, the high-
est proportion of CFSE-labeled lymphocytes
is detected in the peripheral blood and spleen,
with a relatively low percentage of cells being
CFSE+ in the Peyer’s patches and peripheral
lymph nodes. These considerations will influ-
ence the number of events that need to be col-
lected to accurately determine the percentage
of cells that are CFSE+ . Gating on CFSE+
cells and the collection of 5000 events per
fluorescent peak is a convenient means of ac-
quiring information about the CFSE+ lympho-
cytes in cell suspensions, particularly when
there is a low frequency of CFSE+ cells.
Analysis of in vitro–cultured cells. If re-
quired, add a known number of fluorescent
counting beads to the sample prior to collec-
tion on the flow cytometer to allow subse-
quent enumeration of cells in individual CFSE
peaks. Suitable beads are CaliBRITE beads
(Becton-Dickinson) and Flow-Count fluoro-
spheres (Beckman Coulter). When analyzing
the data, apply appropriate electronic gates to
view individually the live cells, apoptotic cells,
and counting beads. The ratio of bead number
to cell number gives the actual cell concentra-
tion in the sample.
For acquisition on the flow cytometer, the
logarithmic mode for the side-scatter (granu-
larity) parameter must be used with counting
beads. Collect all events so that the dataset
includes beads, apoptotic cells, and live cells.
Collect the samples at no more than 700 events
per sec. Collect a sufficient number of events
so that the proportion of cells in each peak can
be accurately determined. For control cultures
where cells have not divided, it is adequate
Current Protocols in Immunology
to collect 5,000 to 10,000 events. For cultures
where cells have divided it is a good idea to col-
lect as many as possible, e.g., 25,000 events.
Comparative studies of CFSE-labeled lym-
phocytes maintained in vitro or in vivo have
shown that the labeled cells have identi-
cal fluorescence-intensity characteristics; i.e,
CFSE-labeled lymphocytes lose the CFSE la-
bel at the same rate whether maintained in vitro
or in vivo (Lyons, 1999).
Fluorescence compensation. Due to the po-
tentially high fluorescence intensity of CFSE-
labeled cells, significant “bleed over” can oc-
cur in those detectors that detect emission
wavelengths close to that of the CFSE/FITC
channel. This is particularly notable in the de-
tectors for PE (or propidium iodide). As such,
it is important to have appropriate unstained
and single-stained cell controls to apply appro-
priate pre- or post-acquisition compensation. It
should also be noted that at CFSE-labeling lev-
els required for detection of maximum cell di-
visions, compensating out CFSE fluorescence
will be extremely difficult within the first 24
hr after labeling and is not recommended. If
examination of the CFSE-labeled cells is re-
quired during this period, the lymphocytes
should be labeled with less CFSE [i.e., one-
quarter (1.25 μM) or one-eighth (0.625 μM)
the normal CFSE-labeling concentration].
Anticipated Results
During the first few days following CFSE
labeling there is rapid decline in CFSE fluo-
rescence, presumably due to the loss of many
CF conjugates from the cells. Despite this loss
in fluorescence, there are sufficient amounts
of long-lived conjugates remaining to allow
nondividing CFSE-labeled lymphocytes to be
tracked in vivo for up to 6 months following
injection. If lymphocyte proliferation is to be
monitored, up to eight divisions can be dis-
cerned for at least a week after cell transfer or
culture initiation. The slow decline in fluores-
cence makes it increasingly difficult to mea-
sure large numbers of cell divisions in ensuing
weeks.
Time Considerations
For a moderate-size experiment, the isola-
tion of lymphocytes, labeling, washing, and
setting up of lymphocyte cultures is accom-
plished in ∼4 hr. Harvesting and washing cells
after culture takes ∼1 hr; an additional 2 to 3 hr
would be required for cell surface or intracel- In Vivo Assays
lular cytokine staining. Collecting cells on the for Lymphocyte
Function
flow cytometer and data analysis take ∼4 hr.
4.9.11
Supplement 84
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Hawkins, E.D., Hommel, M., Turner, M.L., Battye,
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Parish, C.R. 1999. Fluorescent dyes for lymphocyte
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Monitoring lymphocyte proliferation in vitro
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Key References
Gett and Hodgkin, 2000. See above.
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.
Lyons and Parish, 1994. See above.
This is the original paper describing the use of
CFSE to monitor lymphocyte division.
Current Protocols in Immunology
Parish, 1999. See above.
This paper reviews a range of fluorescent dyes for
their use in lymphocyte migration and analysis of
cell division.
Hawkins et al., 2007. See above.
An overview of the different approaches to analyze
CFSE data.

Internet Resources
http://www.vsh.com/
ModFit LT software is an excellent software for
cell proliferation analysis and is available for
Macintosh and PC.
http://www.flowjo.com/
FACS data analysis software, which is currently
available for Macintosh and PC.

In Vivo Assays
for Lymphocyte
Function

4.9.13
Current Protocols in Immunology Supplement 84