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eBiosc
cience™ CFSE
Catalog Number:
N 65-00850
wn as: 5-(and
Also know d 6)-Carboxyfluorescein diacetate succcinimidyl esterr, CFDA SE
For Rese
earch Use Onnly. Not for use in diagno
ostic procedu ures.

Product In
nformation
Contents:
C eBios
science™ CFSSE Formulation: lyophilized
Catalog
C Numbe er: 65-0850 Temperature Limitation: Sto
T ore at -20°C too-
80°C. Protect ffrom light and m
8 moisture. It is
recommended to use the recconstituted dye within
6 months and tto avoid freezee-thawing.
Batch Code: R Refer to vial
Use By: Referr to vial

Descriptio on
CFSE is widely used for cell
c tracking an nd proliferation studies. It has also been use ed in CTL assayys and cell motility
studies. CFFSE readily cro osses intact celll membranes. Once inside th he cells, intrace
ellular esterasees cleave the accetate
groups to yield
y the fluores
scent carboxyfluorescein molecule. The succcinimidyl esterr group reacts with primary am mines,
crosslinking the dye to inttracellular prote
eins. Cell divisiion can be meaasured as succcessive halving g of the fluoresccence
intensity off CFSE. Cells laabeled with CFFSE may be fix xed and permea abilized for ana
alysis of intrace
ellular targets u
using
standard foormaldehyde-containing fixatives and sapon nin-based perm meabilization buuffers, such as the
Foxp3/Tran nscription Factor Staining Bufffer Set (cat. 000-5523) or the IC Fixation Bufffer (cat. 00-82222) and
Permeabilization Buffer (1 10X) (cat. 00-88333).

CFSE has a molecular we eight of 557.47

7. After the aceetate groups are
e cleaved, it ha
as a peak excittation of 494 nmm and
peak emisssion of 521 nm. Each vial of CFSE
C may be reconstituted
r to
o a stock conceentration of 10 mM with 90 μL L of
anhydrous DMSO; once reconstituted
r itt should be use
ed within 6 monnths and proteccted from light and stored at --20°C
with dessic
cant; avoid free
eze-thawing.

ons Reported
Applicatio
CFSE has been reported for use in flow
w cytometric ana
alysis and fluorrescent microsscopy.

Applicatioons Tested
CFSE [5-(aand 6)-carboxyyfluorescein dia
acetate succinim midyl ester] ha s been tested by flow cytomeetric analysis off
stimulated mouse splenocytes. Cells ca an be labeled with
w 0.5 to 20 μM M CFSE. It is rrecommended that the
concentration used for lab
beling cells be carefully deterrmined by eachh investigator fo
or optimal perfo
ormance in thee
assay of interest.

Reference es
Budda SA, Girton A, Henderson JG, Zenewicz LA. Traanscription Facctor HIF-1a Con
ntrols Expressiion of the Cytokine
IL-22 in CD
D4 T Cells. J Im
mmunol. 2016 Oct
O 1;197(7):26
646-52. (CFSE E, FC, PubMed)

Parish CR,, Glidden MH, Quah

Q BJ, Warrren HS. Use off the intracellula
ar fluorescent d
dye CFSE to m
monitor lymphoccyte
a proliferation. Curr Protoc Immunol. 2009
migration and 9 Feb;Chapter 4:Unit4.9.

Ingulli E. Tracing toleranc

ce and immunitty in vivo by CF
FSE-labeling off administered cells. Methodss Mol Biol.
2007;380:3 365-76.

Miller MJ, Wei

W SH, Parker I, Cahalan MD. Two-photon n imaging of lym
mphocyte motillity and antigen
n response in in
ntact
lymph node e. Science. 200
02 Jun 7;296(5
5574):1869-73.

Lyons AB, Parish CR. De

etermination of lymphocyte div
vision by flow ccytometry. J Im
mmunol Method
ds. 1994 May
2;171(1):13
31-7.

Not for further

f distrib
bution withoutt written conseent.
Copyrightt © 2016 Thermo Fisher Scientific Inc. All
A rights rese erved. All trademarks are pproperty of Th
hermo
Fisher Scientiffic and its subbsidiaries unle
ess otherwise e specified.
Tel: 888.999.1371
8 1 or 858.642.2 2058 • Fax x: 858.642.204 46 • thermo ebioscience •
ofisher.com/e
info@e ebioscience.co om
14 March 2017 Rev. 100
 Page
e 2 of 2

eBiosc
cience™ CFSE
Catalog Number:
N 65-00850
wn as: 5-(and
Also know d 6)-Carboxyfluorescein diacetate succcinimidyl esterr, CFDA SE
For Rese
earch Use Onnly. Not for use in diagno
ostic procedu ures.

Related Prroducts
45-0900 CD90.1 (Thy-1.1
1) Monoclonal Antibody
A (HIS551), PerCP-Cyaanine5.5, eBiosscience™ TDS
S DISABLED:
ABMAINT SKU (HIS51)
48-0081 CD8a Monoclonal Antibody (533-6.7), eFluor 450,
4 eBiosciencce™ TDS DISA ABLED: ABMA AINT SKU (53-6
6.7)
Bioscience™ Cell
65-0840 eB C Proliferation Dye eFluor™ ™ 670
65-0842 eB
Bioscience™ Cell
C Proliferation Dye eFluor™ ™ 450

Not for further

f distrib
bution withoutt written conseent.
Copyrightt © 2016 Thermo Fisher Scientific Inc. All
A rights rese erved. All trademarks are pproperty of Th
hermo
Fisher Scientiffic and its subbsidiaries unle
ess otherwise e specified.
Tel: 888.999.1371
8 1 or 858.642.2 2058 • Fax x: 858.642.204 46 • thermo ebioscience •
ofisher.com/e
info@e ebioscience.co om
14 March 2017 Rev. 100
CFSE Cell Labeling Protocol
Protocol: CFSE Cell Labeling
Materials Needed
 PBS
 Flow Cytometry Staining Buffer (cat. 00-4222)

Experimental Procedure
Note: Reconstitute one vial of CFSE to a stock concentration of 10 mM with 90 µL of anhydrous DMSO. Once reconstituted the dye should be used
within 6 months, protected from light, and stored with desiccant at less than or equal to -20°C. Avoid freeze-thawing.
1. Prepare a single-cell suspension of cells to be labeled.
2. Wash cells two times with PBS to remove any serum.
3. Resuspend cells at 5-10x106/mL of PBS (pre-warmed to room temperature).
4. Add CFSE to the desired final concentration (e.g., for a final concentration of 1 µM, add 0.2 µL of a 5 mM stock solution per mL of
cells).
5. Mix immediately and incubate for 10 minutes at room temperature in the dark.
6. Stop labeling by adding 4-5 volumes of cold complete media (containing ≥10% serum) and incubate on ice for 5 minutes.
7. Wash cells 3 times with complete media.
8. Culture or transfer cells, as desired.
Note: The concentration of CFSE, incubation time, and temperature can be modified to achieve the desired staining intensity. However, very high
labeling can lead to compensation issues and may also interfere with cellular functions. Thus, it is highly recommended that each investigator
determine the optimal concentration for the assay of interest.

For Research Use Only. Not for use in diagnostic procedures.

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23 January 2017