Fllow Cytometry

y BestProtocols Page 1 of 10

Stainin
ng Intrace
ellular An
ntigens fo
or Flow C
Cytometry
y
Researc
ch Use Only
y

Protocol A: Two-sttep protocol: intracellular (c

cytoplasmic) p
proteins
Protocol B: One-sttep protocol: intracellular (n
nuclear) prote
eins
Protocol C: Two-sttep protocol: Fixation/Meth
F
hanol

Introduc
ction
A modifica
ation of the ba
asic immunoffluorescent sttaining and flo
ow cytometricc analysis pro
otocol can be used
for the sim
multaneous an
nalysis of surface molecule
es and intrace
ellular antigen
ns at the sing
gle-cell level b
by
flow cytom
metry. Typically, cells are fixed
f
with form
maldehyde to stabilize the cell membran
ne, and then
permeabilized with detergent or alco
ohol to allow antibodies
a
ag
gainst intracelllular antigenss access to sttain
intracellularly.
When staining proteins
s inside the ce
ell, it is importtant to consid
der their location as this ma
ay dictate the
e
protocol and
a buffer sys
stem that will perform optim
mally. For exa
ample, nuclea
ar proteins and many secre
eted
proteins work
w
well with the eBioscience Foxp3/Trranscription F
Factor Stainin
ng Buffer Set (eBioscience Cat.
No. 00-55
523), while secreted protein
ns such as cy
ytokines and cchemokines w
work well with
h the Intracellular
Fixation and
a Permeabilization Buffer Set (eBiosc
cience Cat. No
o. 88-8824). L
Lastly, there a
are several
phosphory
ylated signaling proteins th
hat may not work
w
in the two
o previously-m
mentioned bu
uffer systems but
will work with
w the Fixation/Methanol Protocol. Infformation abo
out performan
nce and prefe
erred buffers is
noted on the
t specific products
p
Tech
hnical Data Sheet. Please contact Tech
hnical Supporrt
(tech@eb
bioscience.com
m) for more in
nformation.

General Notes
1. For op
ptimal perform
mance of fluorrochrome-con
njugated antib
bodies, store vvials at 4C in
n the
dark. Do
D not freeze.
2. Prior to
o use, quickly
y spin the antibody vial to rrecover the m
maximum volu
ume. We do n
not
recommend vortexing the antibo
ody vial.
3. Except where noted
d in the protoc
col, all stainin
ng should be d
done on ice o
or at 4C with
minima
al exposure to
o light.
4. The fix
xation and permeabilization
n steps that a
are required fo
or the detection of intracellular
antigens may alter the
t light scattter propertiess of cells and may increase
e non-specificc
g. Including extra
e
protein ssuch as BSA or FCS in the
e staining bufffer
backgrround staining
may help reduce no
on-specific ba
ackground. W
We also recom
mmend the use
e of the Fixab
ble
Viabilitty Dyes to help eliminate dead
d
cells durring the analyysis.
5. As fixa
ation and perm
meabilization will impact th
he brightness of eFluor nan
nocrystals, we
e
recommend using a minimum fix
xation and pe rmeabilization
n time followe
ed by immediate
analys
sis for optimall results. Som
me generaliza
ations regarding nanocrysttal performancce
performance
after fixation can be
e made, but clone-specific
c
e should be de
etermined
empirically.

Revis
sed 11-25-2015
5
Provided as a co
ourtesy by eBioscience, an Afffymetrix Comp
pany Copyrigh
ht 2000 eBioscience, Inc.
Tel: 888.999..1371 or 858.64
42.2058 Fa
ax: 858.642.20
046 ebioscience.affymetrixx.com
tech@e
ebioscience.co
om

Fllow Cytometry
y BestProtocols Page 2 of 10

Stainin
ng Intrace
ellular An
ntigens fo
or Flow C
Cytometry
y
Researc
ch Use Only
y

Protoco
ol A: Two-s
step protoc
col: intrace
ellular (cyttoplasmic) proteins
The follow
wing protocol allows the sim
multaneous analysis
a
of celll surface molecules and in
ntracellular
antigens at
a the single-c
cell level. In th
his protocol, fixation
f
is follo
owed by perm
meabilization resulting in th
he
creation of
o pores in the
e cell membra
ane that require the continu
uous presencce of the perm
meabilization b
buffer
during all subsequent steps
s
to allow
w antibodies to
o have acces s to the cytop
plasm of the ccell. Thus, all
intracellular staining must be done in the presenc
ce of the perm
meabilization buffer. This p
protocol is
recommended for the detection
d
of cytoplasmic
c
prroteins, cytokkines, or otherr secreted pro
oteins in indivvidual
cells follow
wing activatio
on in vitro or in
n vivo. For cy
ytokine detecction, the apprropriate stimu
ulation conditiions
and kinetics of cytokine
e production will
w vary depe
ending on the cell type and
d the particula
ar cytokine be
eing
assayed. For example,, to stimulate T cells to pro
oduce IFN-, T
TNF-, IL-2, a
and IL-4, a co
ombination off PMA
(a phorbo
ol ester, a prottein kinase C activator) and Ionomycin (a calcium ion
nophore) or a
anti-CD3
antibodies
s can be used
d. To induce IL-6,
I
IL-10, orr TNF- produ
uction by mon
nocytes, stimu
ulation with
lipopolysa
accharide (LP
PS) can be used. For in vittro stimulation
n of cells, it iss necessary to
o block secrettion
of cytokines with protein transport in
nhibitors, such
h as Monensiin or Brefeldin
n A Solution, during the fin
nal
hours of the stimulation
he use and efficacy of diffe
n protocol. It is advised that investigato
ors evaluate th
erent
protein tra
ansport inhibittors in their specific assay system.
For the de
etection of nuclear proteins
s such as tran
nscription facttors, please ssee Protocol B below. Fo
or
detection of some phos
sphorylated signaling
s
mole
ecules such a
as MAPK and STAT protein
ns, it may be
preferential to use Protocol C, belo
ow.
Materrials

12x75 mm round bo
ottom test tub
bes
[Option
nal] Fixable Viability
V
Dyes eFluor 450, 5
506, 660, or 7
780 (eBioscie
ence Cat. No. 650863, 65-0866, 65-0864, 65-086
65)
Directlly conjugated antibodies sp
pecific for intrracellular protteins
Intrace
ellular Fixation
n and Permea
abilization Bu
uffer Set (eBio
oscience Cat.. No. 88-8824
4)
Flow Cytometry
C
Sta
aining Buffer (eBioscience
(
Cat. No. 00-4
4222)
Cell Sttimulation Cocktail (plus prrotein transpo
ort inhibitors) (500X) (eBioscience Cat. No.
00-497
75) or Protein
n Transport In
nhibitor Cockta
ail (500X) (eB
Bioscience Ca
at. No. 00-498
80) or
Brefeld
din A Solution
n (eBioscienc
ce Cat. No. 00
0-4506) or Mo
onensin Soluttion (eBioscie
ence
Cat. No. 00-4505).

Buffe
er and solutio
on preparatio
on
Preparre a 1X working solution off Permeabilizzation Buffer b
by diluting the
e 10X concentrate
with diistilled water prior
p
to use. You will need
d 8.5 mL of Pe
ermeabilizatio
on Buffer for e
each
sample
e.

Experim
mental Proce
edure
1. Preparre cells of inte
erest for evalu
uation of intra
acellular prote
eins. Refer to
o Best Protoco
ols:
Cell Preparation
P
for Flow Cytom
metry.
2. [Option
nal] To elimin
nate potential artifacts due to dead cell ccontamination
n, we recomm
mend
the use of a Fixable
e Viability Dye
e to allow the exclusion of dead cells fro
om the analyssis
(See Best
B
Protocols
s: Staining Dead Cells witth eBioscience
e Fixable Via
ability Dyes
stainin
ng protocol forr instructions for use).

Revis
sed 11-25-2015
5
Provided as a co
ourtesy by eBioscience, an Afffymetrix Comp
pany Copyrigh
ht 2000 eBioscience, Inc.
Tel: 888.999..1371 or 858.64
42.2058 Fa
ax: 858.642.20
046 ebioscience.affymetrixx.com
tech@e
ebioscience.co
om

Fllow Cytometry
y BestProtocols Page 3 of 10

Stainin
ng Intrace
ellular An
ntigens fo
or Flow C
Cytometry
y
Researc
ch Use Only
y
3. Stain cell
c surface antigen(s) as described
d
in B
Best Protocolss for antibodies conjugated
d to
organic fluorochrom
mes: Staining
g cell surface antigens pro
otocol.
4. After th
he last wash, discard the supernatant
s
a
and pulse vorttex the samplle to complete
ely
dissoc
ciate the pellet. Typically ab
bout 100 L rresidual volum
me remains.
5. Fix the
e cells by adding 100 L off IC Fixation B
Buffer and pu
ulse vortex.
6. Incuba
ate tubes in th
he dark at roo
om temperatu re for 20-60 m
minutes.
7. Withou
ut washing, ad
dd 2 mL of 1X
X Permeabilizzation Buffer tto each tube.
8. Centriffuge samples
s at 300-400xg
g at room tem
mperature for 5 minutes, th
hen discard th
he
supern
natant.
9. Resus
spend the cell pellet in 2 mL of 1X Perm
meabilization B
Buffer.
10. Centriffuge samples
s at 300-400xg
g at room tem
mperature for 5 minutes, th
hen discard th
he
supern
natant.
11. Resus
spend the cells in 100 L of
o 1X Permeab
bilization Bufffer. Add the re
ecommended
d
amoun
nt of fluorochrrome-labeled antibody for detection of intracellular antigen(s) to ccells
and incubate in the dark at room
m temperature
e for 20-60 minutes.
12. Add 2 mL of 1X Perrmeabilization
n Buffer to ea
ach tube.
13. Centriffuge samples
s at 300-400xg
g at room tem
mperature for 5 minutes, th
hen discard th
he
supern
natant.
14. Add 2 mL of Flow Cytometry
C
Sta
aining Buffer tto each tube.
15. Centriffuge samples
s at 300-400xg
g at room tem
mperature for 5 minutes, th
hen discard th
he
supern
natant.
16. Resus
spend stained
d cells in an ap
ppropriate vo
olume of Flow
w Cytometry S
Staining Buffer and
acquire samples on
n a flow cytom
meter.

Experim
mental Proce
edure in 96 Well Plate
1.
2.

3.
4.
5.
6.
7.
8.
9.
10.
11.
12.

Prepa
are cells of intterest for evalluation of intra
acellular prote
eins. Refer to
o Best Protoccols:
Cell Preparation
P
for Flow Cytom
metry.
[Option
nal] To elimin
nate potential artifacts due to dead cell ccontamination
n, we recomm
mend
the use of a Fixable
e Viability Dye
e to allow the exclusion of dead cells fro
om the analyssis
(See Best
B
Protocols
s: Protocol C:: Staining De
ead Cells with
h eBioscience
e Fixable Viab
bility
Dyes staining proto
ocol for instructions for use
e).
Stain cell
c surface antigen(s) as described
d
in B
Best Protocolss for antibodies conjugated
d to
organic fluorochrom
mes: Staining
g cell surface antigens pro
otocol.
After th
he last wash, discard the supernatant
s
a
and pulse vorttex the samplle to complete
ely
dissoc
ciate the pellet.
Add 20
00 L of IC Fiixation Bufferr to each well.. It is ideal to add the soluttion such thatt the
cells are
a fully resuspended in the
e solution. Pip
petting is an o
option.
Incuba
ate in the dark
k at room tem
mperature for 3
30-60 minute
es.
Centriffuge samples
s at 300-400xg
g at room tem
mperature for 5 minutes, th
hen discard th
he
supern
natant.
Add 200
2 L 1X Perrmeabilization
n Buffer to ea
ach well.
Centriffuge samples
s at 300-400xg
g at room tem
mperature for 5 minutes, th
hen discard th
he
supern
natant.
Repea
at Steps 8-9.
Resus
spend pellet in
n residual volu
ume and adju
ust volume to about 100 L
L with 1X
Perme
eabilization Bu
uffer.
[Optio
onal] Block with 2% normal mouse/rat s erum by addiing 2 L direcctly to the cells.
Incuba
ate at room te
emperature fo
or 15 minutes..

Revis
sed 11-25-2015
5
Provided as a co
ourtesy by eBioscience, an Afffymetrix Comp
pany Copyrigh
ht 2000 eBioscience, Inc.
Tel: 888.999..1371 or 858.64
42.2058 Fa
ax: 858.642.20
046 ebioscience.affymetrixx.com
tech@e
ebioscience.co
om

Fllow Cytometry
y BestProtocols Page 4 of 10

Stainin
ng Intrace
ellular An
ntigens fo
or Flow C
Cytometry
y
Researc
ch Use Only
y
13. Withou
ut washing, ad
dd the recom
mmended amo
ount of fluorocchrome-conju
ugated antibod
dy for
detection of intracellular antigen((s) to cells an
nd incubate in
n the dark at rroom tempera
ature
for at least 30 minutes.
14. Add 20
00 L of 1X Permeabilizati
P
ion Buffer to e
each well.
15. Centriffuge samples
s at 300-400xg
g at room tem
mperature for 5 minutes, th
hen discard th
he
supern
natant.
16. Repea
at Steps 14-15
5.
17. Resus
spend stained
d cells in an ap
ppropriate vo
olume of Flow
w Cytometry S
Staining Buffer and
acquire samples on
n a flow cytom
meter.

Revis
sed 11-25-2015
5
Provided as a co
ourtesy by eBioscience, an Afffymetrix Comp
pany Copyrigh
ht 2000 eBioscience, Inc.
Tel: 888.999..1371 or 858.64
42.2058 Fa
ax: 858.642.20
046 ebioscience.affymetrixx.com
tech@e
ebioscience.co
om

Fllow Cytometry
y BestProtocols Page 5 of 10

Stainin
ng Intrace
ellular An
ntigens fo
or Flow C
Cytometry
y
Researc
ch Use Only
y

Protoco
ol B: One-s
step protoc
col: intrace
ellular (nuc
clear) prote
eins
The follow
wing protocol allows the sim
multaneous analysis
a
of celll surface molecules and in
ntracellular
antigens, including nuc
clear antigens
s, at the single
e-cell level. T
This protocol ccombines fixa
ation and
permeabilization into a single step. This
T
protocol is recommen
nded for the d
detection of nu
uclear antigen
ns
such as trranscription fa
actors but is also
a
useful forr the detectio n of many cyttokines. For ccompatibility o
of the
Foxp3/Tra
anscription Fa
actor Staining
g Buffer Set (e
eBioscience C
Cat. No. 00-5523) with cyto
okine antibod
dies,
please se
ee our Buffer Compatibility
C
chart online: http://www.eb
bioscience.co
om/resourcess/application/fflowcytometry
y/antibody-fixa
ation-considerations.htm.
Materrials

12x75 mm round bo
ottom test tub
bes or 96 welll V or U botto
om plate
[Option
nal] Fixable Viability
V
Dyes eFluor 450, 5
506, 660 and 780 (eBioscience Cat. No
o. 650863, 65-0866, 65-0864, 65-086
65)
[Option
nal] Normal Mouse
M
Serum
m (eBioscience
e Cat. No. 24-5544)
[Option
nal] Normal Rat
R Serum (eB
Bioscience Ca
at. No. 24-5555)
Directlly conjugated antibodies sp
pecific for intrracellular protteins
Foxp3/Transcription
n Factor Stain
ning Buffer Se
et (eBiosciencce Cat. No. 00-5523)
Flow Cytometry
C
Sta
aining Buffer (eBioscience
(
Cat. No. 00-4
4222)

Buffe
ers and soluttion preparattion
Preparre fresh Foxp
p3 Fixation/Pe
ermeabilizatio
on working so
olution by dilutting Foxp3
Fixatio
on/Permeabilization Conce
entrate (1 partt) with Foxp3 Fixation/Perm
meabilization
Diluen
nt (3 parts). You
Y will need 1 mL of the F
Fixation/Permeabilization w
working solutio
on for
each sample.
s
Preparre a 1X working solution off Permeabilizzation Buffer b
by diluting the
e 10X concentrate
with diistilled water prior
p
to use. You
Y will need 8.5 mL of Pe
ermeabilizatio
on Buffer for e
each
sample
e, if staining in tubes.

Experim
mental Proce
edure in tub
bes
1. Preparre cells of inte
erest for evalu
uation of intra
acellular prote
eins. Refer to
o Best Protoco
ols:
Cell Preparation
P
for Flow Cytom
metry.
2. [Option
nal] To elimin
nate potential artifacts due to dead cell ccontamination
n, we recomm
mend
the use of a Fixable
e Viability Dye
e to allow the exclusion of dead cells fro
om the analyssis
B
Protocols
s: Protocol C:: Staining De
ead Cells with
h eBioscience
e Fixable Viab
bility
(See Best
Dyes staining proto
ocol for instructions for use
e).
3. Stain cell
c surface antigen(s) as described
d
in B
Best Protocolss for antibodies conjugated
d to
organic fluorochrom
mes: Staining
g cell surface antigens pro
otocol.
4. After th
he last wash, discard the supernatant
s
a
and pulse vorttex the samplle to complete
ely
dissoc
ciate the pellet.
5. Add 1 mL of Foxp3 Fixation/Perm
meabilization working solu
ution to each ttube and pulsse
vortex.
6. Incuba
ate at in the dark at 4C or room temperrature for 30-6
60 minutes. (Mouse samples
can be
e incubated fo
or up to 18 ho
ours at 4C in the dark).
7. Withou
ut washing, ad
dd 2 mL of 1X
X Permeabilizzation Buffer tto each tube.
8. Centriffuge samples
s at 300-400xg
g at room tem
mperature for 5 minutes, th
hen discard th
he
supern
natant.
Revis
sed 11-25-2015
5
Provided as a co
ourtesy by eBioscience, an Afffymetrix Comp
pany Copyrigh
ht 2000 eBioscience, Inc.
Tel: 888.999..1371 or 858.64
42.2058 Fa
ax: 858.642.20
046 ebioscience.affymetrixx.com
tech@e
ebioscience.co
om

Fllow Cytometry
y BestProtocols Page 6 of 10

Stainin
ng Intrace
ellular An
ntigens fo
or Flow C
Cytometry
y
Researc
ch Use Only
y
9. [Optio
onal] Repeat Steps
S
7-8.
10. Resus
spend pellet in
n 100 L of 1X
X Permeabilizzation Buffer. This is typiccally the residual
volume
e after decanting.
11. [Option
nal] Block witth 2% normal mouse/rat se
erum by addin
ng 2 L directtly to the cellss.
Incuba
ate at room te
emperature fo
or 15 minutes..
12. Withou
ut washing, ad
dd the recom
mmended amo
ount of fluorocchrome-conju
ugated antibod
dy for
detection of intracellular antigen((s) to cells an
nd incubate in
n the dark at rroom tempera
ature
for at least 30 minutes.
13. Add 2 mL of 1X Perrmeabilization
n Buffer to ea
ach tube.
14. Centriffuge samples
s at 300-400xg
g at room tem
mperature for 5 minutes, th
hen discard th
he
supern
natant.
15. Add 2 mL of 1X Perrmeabilization
n Buffer or Flo
ow Cytometryy Staining Bufffer to each tu
ube.
16. Centriffuge samples
s at 300-400xg
g at room tem
mperature for 5 minutes, th
hen discard th
he
supern
natant.
17. Resus
spend stained
d cells in an ap
ppropriate vo
olume of Flow
w Cytometry S
Staining Buffer and
acquire samples on
n a flow cytom
meter.

Experim
mental Proce
edure in 96 Well Plate
18. Prepa
are cells of intterest for evalluation of intra
acellular prote
eins. Refer to
o Best Protoccols:
Cell Preparation
P
for Flow Cytom
metry.
19. [Option
nal] To elimin
nate potential artifacts due to dead cell ccontamination
n, we recomm
mend
the use of a Fixable
e Viability Dye
e to allow the exclusion of dead cells fro
om the analyssis
(See Best
B
Protocols
s: Protocol C:: Staining De
ead Cells with
h eBioscience
e Fixable Viab
bility
Dyes staining proto
ocol for instructions for use
e).
20. Stain cell
c surface antigen(s) as described
d
in B
Best Protocolss for antibodies conjugated
d to
organic fluorochrom
mes: Staining
g cell surface antigens pro
otocol.
21. After th
he last wash, discard the supernatant
s
a
and pulse vorttex the samplle to complete
ely
dissoc
ciate the pellet.
22. Add 20
00 L of Foxp
p3 Fixation/Pe
ermeabilizatio
on working so
olution to each
h well. It is ide
eal to
add the solution suc
ch that the ce
ells are fully re
esuspended iin the solution
n. Pipetting iss an
option.
23. Incuba
ate in the dark
k at room tem
mperature for 3
30-60 minute
es. (Mouse sa
amples can be
e
incuba
ated for up to 18 hours at 4C
4 in the darrk).
24. Centriffuge samples
s at 300-400xg
g at room tem
mperature for 5 minutes, th
hen discard th
he
supern
natant.
25. Add 200
2 L 1X Perrmeabilization
n Buffer to ea
ach well.
26. Centriffuge samples
s at 300-400xg
g at room tem
mperature for 5 minutes, th
hen discard th
he
supern
natant.
27. Repea
at Steps 8-9.
28. Resus
spend pellet in
n residual volu
ume and adju
ust volume to about 100 L
L with 1X
Perme
eabilization Bu
uffer.
29. [Optio
onal] Block with 2% normal mouse/rat s erum by addiing 2 L direcctly to the cells.
Incuba
ate at room te
emperature fo
or 15 minutes..
30. Withou
ut washing, ad
dd the recom
mmended amo
ount of fluorocchrome-conju
ugated antibod
dy for
detection of intracellular antigen((s) to cells an
nd incubate in
n the dark at rroom tempera
ature
for at least 30 minutes.
00 L of 1X Permeabilizati
P
ion Buffer to e
each well.
31. Add 20

Revis
sed 11-25-2015
5
Provided as a co
ourtesy by eBioscience, an Afffymetrix Comp
pany Copyrigh
ht 2000 eBioscience, Inc.
Tel: 888.999..1371 or 858.64
42.2058 Fa
ax: 858.642.20
046 ebioscience.affymetrixx.com
tech@e
ebioscience.co
om

Fllow Cytometry
y BestProtocols Page 7 of 10

Stainin
ng Intrace
ellular An
ntigens fo
or Flow C
Cytometry
y
Researc
ch Use Only
y
32. Centriffuge samples
s at 300-400xg
g at room tem
mperature for 5 minutes, th
hen discard th
he
supern
natant.
33. Add 20
00 L of 1X Permeabilizati
P
ion Buffer or F
Flow Cytome
etry Staining B
Buffer to each
h well.
34. Centriffuge samples
s at 300-400xg
g at room tem
mperature for 5 minutes, th
hen discard th
he
supern
natant.
35. Resus
spend stained
d cells in an ap
ppropriate vo
olume of Flow
w Cytometry S
Staining Buffer and
acquire samples on
n a flow cytom
meter.

Revis
sed 11-25-2015
5
Provided as a co
ourtesy by eBioscience, an Afffymetrix Comp
pany Copyrigh
ht 2000 eBioscience, Inc.
Tel: 888.999..1371 or 858.64
42.2058 Fa
ax: 858.642.20
046 ebioscience.affymetrixx.com
tech@e
ebioscience.co
om

Fllow Cytometry
y BestProtocols Page 8 of 10

Stainin
ng Intrace
ellular An
ntigens fo
or Flow C
Cytometry
y
Researc
ch Use Only
y

Protoco
ol C: Two-s
step protoc
col: Fixatio
on/Methano
ol
The follow
wing protocol allows for the
e simultaneou
us analysis off cell surface m
molecules an
nd some
intracellular phosphory
ylated signalin
ng proteins. In this protoco
ol, fixation is ffollowed by trreatment of ce
ells
with Methanol. For pho
ospho-protein
n detection, th
he appropriate
e stimulation conditions an
nd kinetics of
phosphory
ylation will va
ary depending
g on the cell ty
ype and the p
particular sign
naling event b
being assayed
d.
For example, to induce
e phospho-ST
TAT1 (Y701) phosphorylati
p
ion, macrophages can be activated with
h
IFN or IF
FN, while phospho-ERK1//2 (T202/Y204) is induced in T cells in rresponse to P
PMA (a phorb
bol
ester, a protein kinase C activator) or
o CD3 antibo
odies.

Gene
eral Notes
1. Fluorochrome--conjugated antibodies
a
can
n be used to sstain surface proteins for the purpose o
of
im
mmunophenotyping cells th
hat will be furtther analyzed
d for phospho
orylated proteins, however,,
ad
dditional cons
siderations for staining are
e warranted.

Antibo
ody staining fo
or surface ma
arkers on live cells has bee
en shown to a
alter expressio
on of
signaliing proteins due
d to possible stimulation//suppression of signaling e
events. Beca
ause
prior to cell sttimulation. In
of this,, surface stain
ning is not rec
commended p
nstead, stain
surface proteins at the same step as the intra
acellular prote
ein staining. P
Please note th
hat
some proteins will also
a
have intracellular poolls, in addition
n to surface lo
ocalization, wh
hich
should
d be considere
ed. Antibody clones to surf
rface proteins that will reco
ognize fixed
cells/e
epitopes will need
n
to be eva
aluated and u
used. Refer to
o
http://u
us.ebioscienc
ce.com/resourrces/applicati on/flow-cytom
metry/clone-performance-a
afterfix-perrm.htm in Tec
chnical Suppo
ort Resourcess.

If surfa
ace staining is
s required prior to the fixattion step in Sttep 5 (due to epitope
destruction), cells may
m be staine
ed with fluorocchrome-conju
ugated antibod
dies before th
he
Fixatio
on/MeOH step
ps only if the conjugated
c
flu
uorochromess are resistantt to methanol
exposu
ure.
Me
eOH Resistantt Fluorochrom
mes
Ale
exa Fluor 488
eF
Fluor 660
Ale
exa Fluor 647
eF
Fluor 450
FIT
TC

Me
eOH Sensitive
e Fluorochrom
mes
PE
E
PE
E-tandems
Pe
erCP
Pe
erCP-tandems
AP
PC
AP
PC-tandems

2. For adherent cells,

c
we recommend fixing
g the cells (Sttep 5) in the p
plates/well. A
After fixation,
crape cells orr treat with ED
DTA solution to
t harvest an d continue wiith protocol. T
Trypsin can be
sc
e
us
sed if you are
e not staining for surface antibodies or yyou know you
ur surface pro
otein is resista
ant to
tryspin digestio
on.

Revis
sed 11-25-2015
5
Provided as a co
ourtesy by eBioscience, an Afffymetrix Comp
pany Copyrigh
ht 2000 eBioscience, Inc.
Tel: 888.999..1371 or 858.64
42.2058 Fa
ax: 858.642.20
046 ebioscience.affymetrixx.com
tech@e
ebioscience.co
om

Fllow Cytometry
y BestProtocols Page 9 of 10

Stainin
ng Intrace
ellular An
ntigens fo
or Flow C
Cytometry
y
Researc
ch Use Only
y
Materrials

12
2x75 mm roun
nd bottom test tubes or 96--well round orr V- bottom m
microtiter plate
es
Prrimary antibod
dies (directly conjugated)
c
eB
Bioscience Flo
ow Cytometry
y Staining Bufffer (Cat. No. 00-4222)
eB
Bioscience IC Fixation Bufffer (cat. 00-82
222)
90
0-100% Metha
anol (HPLC grade)
g
Op
ptional Fc Blo
ock: Anti-Mouse CD16/CD3
32 Purified (e
eBioscience C
Cat. No. 14-01
161)
or Human Fc Receptor
R
Binding Inhibitor P
Purified (eBio
oscience Cat. No. 14-9161))

Experim
mental Proce
edure
1. Preparre cells of inte
erest for stimu
ulation in app
propriate med ia.
2. Count cells and res
suspend in ap
ppropriate me dia at ~1-5x106 cells/mL.
3. Stimulate cells at 37
7C with apprropriate treatm
ment for desirred time point(s). Remem
mber
to incu
ubate untreate
ed cells at 37C as a nega
ative control.
4. [Option
nal] If surface
e staining is needed prior to
o fixation (in S
Step 5), stain
n cell surface
antigen(s) as descrribed in Best Protocols
P
Sta
aining Cell Su
urface Antigen
ns for antibod
dies
conjug
gated to methanol-resistant fluorochrom
mes.
5. At the end of the stiimulation periiod, fix cells to
o stop stimula
ation by addin
ng an equal
volume
e of IC Fixatio
on Buffer dire
ectly to cells a
and vortex.
6. Incuba
ate cells in the
e dark at room
m temperature
e for 10-60 m
minutes.
7. Centriffuge cells at 600xg
6
at room
m temperature
e for 4-5 minu
utes, then disscard superna
atant.
8. Resus
spend the cell pellet in resid
dual volume a
and add 1 mL
L ice-cold 90--100% methanol,
vortex, and incubate at 4C or on
n ice for at lea
ast 30 minute
es.
E: Once in methanol, cells can
c be stored
d at <-20C fo
or up to 4 wee
eks.
NOTE
9. Wash cells with an excess volum
me of Flow Cyytometry Stain
ning Buffer.
10. Centriffuge cells at 600xg
6
at room
m temperature
e for 4-5 minu
utes, then disscard superna
atant.
11. Resus
spend cells at 1x107 cells/m
mL in Flow Cyytometry Stain
ning Buffer.
12. Aliquot 1x106 cells (100 L) into separate flow
w tubes.
13. [Option
nal] Cells can
n be blocked for
f nonspecifiic Fc-mediate
ed binding usiing Anti-Mousse
CD16//CD32 Purified or Human Fc
F Receptor B
Binding Inhibiitor Purified p
prior to stainin
ng.
14. Add th
he recommended amount of
o fluorochrom
me-labeled an
ntibody to eacch tube and
incuba
ate in the dark
k at room tem
mperature for 3
30-60 minutes.
NOTE
E: If needed, surface
s
stainin
ng and intrace
ellular phosph
ho staining ca
an be perform
med
simulta
aneously. As
s not all antibo
ody clones wiill bind to a fixxed epitope, p
please refer to
o
online table for antiibody clones that
t
will stain cells after fixxation and me
ethanol treatm
ment.
15. Wash cells with 2 mL
m Flow Cytom
metry Stainin g Buffer and
d centrifuge att 600xg at roo
om
temperature for 4-5
5 minutes. Repeat step 15.
16. Resus
spend stained
d cells in an ap
ppropriate vo
olume of Flow
w Cytometry S
Staining Buffer and
acquire samples on
n a flow cytom
meter.

Experim
mental Proce
edure in 96 Well Plate
1. Preparre cells of inte
erest for stimu
ulation in app
propriate med ia.
2. Count cells and res
suspend in ap
ppropriate me dia at ~1-5x106 cells/mL.
3. Add 10
00 L approp
priate treatmen
nt to wells in a 96 well platte.

Revis
sed 11-25-2015
5
Provided as a co
ourtesy by eBioscience, an Afffymetrix Comp
pany Copyrigh
ht 2000 eBioscience, Inc.
Tel: 888.999..1371 or 858.64
42.2058 Fa
ax: 858.642.20
046 ebioscience.affymetrixx.com
tech@e
ebioscience.co
om

Flo
ow Cytometry BestProtocols Page 10
0 of 10

Stainin
ng Intrace
ellular An
ntigens fo
or Flow C
Cytometry
y
Researc
ch Use Only
y
4. Add 10
00 L cells to
o wells and stimulate cells a
at 37C for de
esired time po
oint(s).
Remem
mber to incub
bate untreated
d cells at 37C
C as a negatiive control.
5. [Option
nal] If surface
e staining is needed prior to
o fixation (in S
Step 5), stain
n cell surface
antigen(s) as descrribed in Best Protocols
P
Sta
aining Cell Su
urface Antigen
ns for antibod
dies
conjug
gated to methanol resistantt fluorochrom
mes.
6. At the end of the stiimulation periiod, fix cells to
o stop stimula
ation by addin
ng 200 L of IC
Fixatio
on Buffer direc
ctly to wells.
7. Incuba
ate plate in the dark at room
m temperaturre for 10-60 m
minutes.
8. Centriffuge plate at 600xg at room
m temperaturre for 4-5 minutes, then disscard superna
atant.
9. Resus
spend the cell pellets in res
sidual volume
e and add 100
0 L ice-cold 90-100%
methanol to wells, vortex,
v
and in
ncubate plate at 4C or on ice for at leasst 30 minutes.
E: Once in methanol, cells can
c be stored
d at <-20C fo
or up to 4 wee
eks.
NOTE
10. Wash cells with 200
0 L Flow Cyttometry Stain
ning Buffer.
11. Centriffuge cells at 600xg
6
at room
m temperature
e for 4-5 minu
utes, then disscard superna
atant.
12. Repea
at steps 10 an
nd 11.
13. [Optio
onal] Cells can
n be blocked for nonspeciffic Fc-mediate
ed binding ussing Anti-Mouse
CD16//CD32 Purified or Human Fc
F Receptor B
Binding Inhibiitor Purified p
prior to stainin
ng.
14. Add th
he recommended amount of
o fluorochrom
me-labeled an
ntibody to eacch well and
incuba
ate for in the dark
d
at room temperature
t
3
30-60 minutes.
NOTE
E: If needed, surface
s
stainin
ng and intrace
ellular phosph
ho staining ca
an be perform
med
simulta
aneously. As
s not all antibo
ody clones wiill bind to a fixxed epitope, p
please refer to
o
online table for antiibody clones that
t
will stain cells after fixxation and me
ethanol treatm
ment.
15. Wash wells with 200 L Flow Cy
ytometry Stain
ning Buffer a
and centrifuge
e at 600xg for 4-5
minute
es. Repeat ste
ep 15.
16. Resus
spend stained
d cells in an ap
ppropriate vo
olume of Flow
w Cytometry S
Staining Buffer and
acquire samples on
n a flow cytom
meter.

Revis
sed 11-25-2015
5
Provided as a co
ourtesy by eBioscience, an Afffymetrix Comp
pany Copyrigh
ht 2000 eBioscience, Inc.
Tel: 888.999..1371 or 858.64
42.2058 Fa
ax: 858.642.20
046 ebioscience.affymetrixx.com
tech@e
ebioscience.co
om