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Improved methods have continually been
sought to analyze single-cell cytokine production by human and rodent
leukocytes and cell lines. Single-cell cytokine analysis techniques
include ELISPOT, in situ hybridization, immunohistochemistry, limiting
dilution analysis and single cell PCR.1 These
latter techniques have significant drawbacks requiring either high
technical proficiency or tedious data collection and analysis. Flow
cytometry is a powerful analytical technique in which individual cells
can be simultaneously analyzed for several parameters, including size
and granularity, as well as the expression of surface and intracellular
markers defined by fluorescent antibodies.1-5
Recently, fluorescent anti-cytokine and
anti-chemokine monoclonal antibodies have become very useful for the
intracellular staining and multiparameter flow cytometric analysis of
individual cytokine-producing cells within mixed cell populations.1,3-8 Multicolor immunofluorescent staining with
antibodies against intracellular cytokines and cell surface markers
provides a high resolution method to identify the nature and frequency
of cells which express a particular cytokine(s). For example,
multicolor immunofluorescent staining of an individual cell surface
antigen and two cytoplasmic cytokines has been used to identify and
enumerate cell types which express cytokines in either a restricted (e.g.,
Th1- versus Th2-like cells) or unrestricted (e.g., Th0-like
cells) pattern . 9,10 In addition to enabling highly specific and
sensitive measurements of several parameters for individual cells
simultaneously, this method has the capacity for rapid analysis of
large numbers of cells which are required for making statistically
significant measurements.2
Staining of intracellular cytokines
depends on the identification of cytokine-specific monoclonal
antibodies which are compatible with a fixation-permeabilization
procedure.11-13 Optimal intracellular cytokine staining has been
reported using a combination of fixation with paraformaldehyde and
subsequent permeabilization of cell membranes with the detergent
saponin. Paraformaldehyde fixation allows preservation of cell
morphology and intracellular antigenicity, while also enabling the
cells to withstand permeabilization by detergent. Membrane
permeabilization by saponin allows the cytokine-specific monoclonal
antibody to penetrate the cell membrane, cytosol, and membranes of the
endoplasmic reticulum and Golgi apparatus.
Critical parameters for cytokine staining
include the following: cell type and activation protocol; the time of
cell harvest following activation; the inclusion of a protein transport
inhibitor during cell activation; and the choice of anti-cytokine
antibody.
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A.
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Stimulation of Cells
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Various in vitro methods have been reported for stimulating
cytokine producing cells.1,3-15 Polyclonal activators
have been particularly useful for inducing and characterizing cytokine-producing
cells. These activators include: phorbol esters plus calcium ionophore;
phytohemaglutinin; Staphylococcus enterotoxin B; and monoclonal
antibodies directed against subunits of the TCR/CD3 complex (with
or without antibodies directed against costimulatory receptors such
as CD28).
NOTE: It has been reported that cell
activation with PMA alone causes a transient loss of CD4 expression
from the surface of mouse T cells. Cell activation with PMA and calcium
ionophore together has been reported to cause a greater and more
sustained decrease in CD4 expression as well as a decrease in CD8
expression in mouse thymocytes and mouse and human peripheral T
lymphocytes.8
BDB-Pharmingen recommends the use of an intracellular protein
transport inhibitor during in vitro cell activation for cytokine
staining. Use of GolgiStop™ (cat. no. 554724; containing monensin)
or GolgiPlug™ (cat. no. 555029; containing brefeldin A) block intracellular
transport processes and results in the accumulation of most cytokine
proteins in the rough endoplasmic reticulum or Golgi complex. This
leads to an enhanced ability to detect cytokine-producing cells.
Since these agents have a dose- and time-dependent cytotoxic effect,
exposure must be limited.
NOTE: Investigators should be aware of
possible effects of transport inhibitors on the expression levels of
cell surface markers. Brefeldin
A has been found to cause decreased levels of CD14 staining.
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1.
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Cultures for Generating Human
Cytokine-Producing Cells
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a.
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IL-3+ , IL-4+ , IL-5+
, IL-13+ and GM-CSF+ human cells:
Human PBMC, purified human CD4+ or CD8+
cells (especially for IL-5+ and IL-13+
cells) are stimulated with immobilized anti-human CD3
antibody (clone UCHT1, 10 µg/ml for plate coating, cat.
no. 555329), soluble anti-human CD28 antibody (clone
CD28.2, 2 µg/ml; cat. no. 555725), recombinant human
IL-2 (10 ng/ml; cat. no. 554603) and recombinant human
IL-4 (20 ng/ml; cat. no. 554605) for 2 days. The cells
are washed and subsequently cultured in medium containing
rhIL-2 and rhIL-4 for another 3 days. Finally, the cells
are harvested and restimulated for 4 hr with PMA (5
ng/ml; Sigma, cat. no. P-8139), calcium ionophore A23187
(250 ng/ml; Sigma, cat. no. C-9275), or ionomycin (500
ng/ml; Sigma, cat. no. I-0634) in the presence of a
protein transport inhibitor (either GolgiStop, cat.
no. 554724, or GolgiPlug, cat. no. 555029).
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b.
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TNF- α+ human cells: Human PBMC are
stimulated with immobilized anti-human CD3 antibody
(clone UCHT1, 10 µg/ml for plate coating, cat. no. 555329)
and recombinant human IL-2 (10 ng/ml; cat. no. 554603)
for 2 days. The cells are washed and subsequently cultured
in medium containing rhIL-2 for another 3 days. Finally,
the cells are harvested and restimulated for 4 hr with
PMA (5 ng/ml; Sigma, cat. no. P-8139), calcium ionophore
A23187 (250 ng/ml; Sigma, cat. no. C-9275), or ionomycin
(500 ng/ml; Sigma, cat. no. I-0634) or alternatively
the cells can be restimulated with anti-CD3 and anti-CD28.
Restimulation should be performed in the presence of
a protein transport inhibitor (either GolgiStop, cat.
no. 554724, or GolgiPlug, cat. no. 555029).
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c.
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IL-2+ , TNF- α+ , and IFN- γ
++ human cells: Human PBMC are stimulated
for 6 hr with PMA (5 ng/ml; Sigma, cat. no. P-8139),
calcium ionophore A23187 (500 ng/ml; Sigma, cat. no.
C-9275), or ionomycin (500 ng/ml; Sigma cat. no. I-0634)
in the presence of a protein transport inhibitor (either
GolgiStop, cat. no. 554724, or GolgiPlug, cat. no. 555029).
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d.
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IL-1α + , IL-6+ , IL-8+
and GRO- α+ human cells: Human PBMC
are stimulated for 4 hr with LPS (1.0 µg/ml; Sigma cat.
no. L-8274) in the presence of a protein transport inhibitor
(either GolgiStop, cat. no. 554724, or GolgiPlug, cat.
no. 555029).
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e.
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IL-10+ , MCP-1+ , MIP-1 α+
, MCP-3+ , and MIG+ human cells:
Human PBMC are stimulated for 24 hr with LPS (1.0 µg/ml)
in the presence of a protein transport inhibitor (either
GolgiStop, cat. no. 554724, or GolgiPlug, cat. no. 555029).
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f.
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IL-12+ human cells: Human PBMC are primed
for 2 hr with rhIFN- γ (10 ng/ml; cat. no. 554616)
and are then activated with IFN- γ (10 ng/ml) and
LPS (1.0 µg/ml; Sigma, cat. no. L-8274) in the presence
of a protein transport inhibitor (either GolgiStop,
cat. no. 554724, or GolgiPlug, cat. no. 555029) for
an additional 22 hr.
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g.
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RANTES+ human cells: Because T cells can
make RANTES constitutively (although its expression
is upregulated by cell stimulation), human PBMC can
simply be cultured for 24 hr in the presence of a protein
transport inhibitor (GolgiStop™ is preferred, cat. no.
554724).
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2.
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Cultures for Generating Mouse
Cytokine-Producing Cells
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a.
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IL-2+ , TNF- α+ , and IFN-
γ+ mouse cells: Mouse splenocytes are
stimulated for 4 hr with PMA (5 ng/ml; Sigma, cat. no.
P-8139) and ionomycin (500 ng/ml; Sigma, cat. no. I-0634)
in the presence of a protein transport inhibitor (either
GolgiStop, cat. no. 554724, or GolgiPlug, cat. no. 555029).
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b.
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IL-3+ , IL-4+ , IL-5+
, IL-10+ , GM-CSF+ mouse cells:
Purified CD4+ mouse splenocytes from 6-month
old BALB/c mice are stimulated with plate-bound anti-mouse
CD3 (clone 145-2C11, 25 µg/ml; cat. no. 553057) and
soluble anti-mouse CD28 (clone 37.51, 2 µg/ml; cat.
no. 553294) for 2 days in culture together with rmIL-2
(10 ng/ml; cat. no. 550069) and rmIL-4 (50 ng/ml; cat.
no. 550067), followed by a 3 day incubation with only
rmIL-2 and rmIL-4. This is followed by a 4 hr stimulation
with plate-bound anti-mouse CD3 (25 µg/ml) and anti-mouse
CD28 (2 µg/ml) in the presence of a protein transport
inhibitor. Alternatively, can restimulate with PMA (5
ng/ml; Sigma, cat. no. P-8139) and ionomycin (500 ng/ml;
Sigma, cat. no. I-0634) in the presence of a protein
transport inhibitor (either GolgiStop, cat. no. 554724,
or GolgiPlug, cat. no. 555029).
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c.
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IL-6+ , IL-12+ , TNF- α+
mouse cells: 3-day thioglycolate elicited peritoneal
cells were harvested and stimulated with 1 µg/ml LPS
and GolgiPlug™ for 4 hr.
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d.
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MCP-1+ mouse cells: Thioglycolate-elicited
peritoneal macrophages from 6-month old BALB/c mice
are stimulated with LPS (1 µg/ml; Sigma, cat. no. L-8274)
overnight in the presence of a protein transport inhibitor
(either GolgiStop, cat. no. 554724, or GolgiPlug, cat.
no. 555029).
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3.
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Cultures for Generating Rat
Cytokine-Producing Cells
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a.
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IL-4+ and IL-10+ rat cells:
Purified splenic CD4+ cells from an adult
rat are stimulated with plate-bound anti-rat CD3 (clone
G4.18, 25 µg/ml; cat. no.. 554829) and soluble anti-rat
CD28 (clone JJ319, 2 µg/ml; cat. no. 554993) for 2 days
in culture together with recombinant rat IL-2 (10 ng/ml;
cat. no. 555106) and rrIL-4 (50 ng/ml; cat. no. 555107),
followed by a 3 day incubation with only rrIL-2 and
rrIL-4. This is followed by a 4-6 hr stimulation with
PMA (5 ng/ml; Sigma, cat. no. P-8139) and ionomycin
(500 ng/ml; Sigma, cat. no. I-0634) in the presence
of a protein transport inhibitor. Alternatively, can
restimulate with plate-bound anti-rat CD3 and soluble
anti-rat CD28 for 4-6 hr in the presence of a protein
transport inhibitor (either GolgiStop, cat. no. 554724,
or GolgiPlug, cat. no. 555029).
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B.
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Protocol: Multicolor Staining for
Intracellular Cytokines and Cell Surface Antigens
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1.
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Harvest Cells
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Viable activated cell populations can be prepared from in
vivo-stimulated tissues or from in vitro- stimulatory
cultures. The cells can be suspended and distributed to plastic
tubes or microwell plates for immunofluorescent staining.
Cells should be protected from light throughout staining and
storage.
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2.
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Block Fc Receptors
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Reagents that block Fc receptors may be
useful for reducing nonspecific immunofluorescent staining.14
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a.
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In the mouse system, purified 2.4G2 antibody directed
against FcgII/III receptors
(Fc Block™; cat. no. 553141 and 553142), can be used
to block nonspecific staining by fluorochrome conjugated
antibodies which is mediated by receptors. To block
mouse Fc receptors with Mouse Fc Block™, preincubate
cell suspension with 1 µg Fc Block™ per 106
cells in 100 µl of Staining Buffer for 15 min at 4°C.
The cells are then stained with a fluorescent antibody
which is specific for a cell surface antigen of interest.
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b.
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Fc receptors on human cells can be pre-blocked by
incubating cells with an excess of irrelevant purified
Ig from the same species and with the same isotype as
the antibodies used for immunofluorescent staining or
by incubation with 10% FCS in PBS.
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c.
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In the rat system, purified D34-485 antibody directed
against FcgII receptor (Rat
BD Fc Block™; cat. no. 550270 and 550271), can be used
to block nonspecific staining by fluorochrome conjugated
antibodies which is mediated by receptors. To block
rat Fc receptors with Rat Fc Block™, preincubate cell
suspension with 1 µg Fc Block™ per 106 cells
in 100 µl of Staining Buffer for 15 min at 4°C. The
cells are then stained with a fluorescent antibody which
is specific for a cell surface antigen of interest.
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3.
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Stain Cell Surface Antigens
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a.
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Incubate ~106 cells in 50 µl of Staining Buffer (100 µl for staining
in tubes) with a pre-titrated optimal concentration (≤0.5 µg) of a fluorochrome-conjugated monoclonal
antibody specific for a cell surface antigen, such as, CD3, CD4,
CD8, CD14, or CD19 (15-30 min, 4°C). Multicolor staining of
different cell surface antigens can be carried out to provide
controls for setting proper compensation of the brightest
fluorescent signals.
NOTE:Some antibodies which
recognize native cell surface markers may not bind to
fixed/denatured antigen. For this reason, it is recommended that
the staining of cell surface antigens be done with live, unfixed
cells PRIOR to fixation/permeabilization and staining of
intracellular cytokines.
Altering the procedure such that cells
are fixed prior to staining of cell surface antigens requires that
suitable antibody clones be empirically identified.
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b.
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Wash cells 2X with Staining Buffer (1
ml/wash for staining in tubes), pellet by centrifugation (250 X g),
and remove supernatant.
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4.
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Fix and Permeabilize Cells
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a.
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Thoroughly resuspend cells in 100 µl
(250 µl for staining in tubes) of Cytofix/Cytoperm™ solution for
10-20 min at 4°C.
NOTE: Cell aggregation can be
avoided by vortexing prior to the addition of the Cytofix/Cytoperm™
solution.
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b.
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Wash cells two times in 1X Perm/Wash™
solution (1 ml/wash for staining in tubes), pellet, and remove
supernatant.
NOTE: Perm/Wash ™ solution is
required in washing steps to maintain cells in a permeabilized
state.
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5.
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Alternative Fixation and
Permeabilization Protocol
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Cells can be fixed and stored to
continue the intracellular staining at a later time.
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a.
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Fixation and Storage of Cells
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1.
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Resuspend cells in 100 µl (250 µl
for tubes or 1 ml/107 cells for
bulk fixing) of a 4% paraformaldehyde solution at 4°C for 10-20
min.
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2.
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Wash cells 2X in staining buffer
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3.
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Resuspend cells in staining buffer for storing cells at 4°C
or in 90% FCS/10% DMSO for storing at -80°C
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b.
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Permeabilizing Fixed Cells
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1.
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For frozen cells, wash 2X to remove
DMSO.
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2.
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Resuspend cells in Perm/Wash™ for 15
min
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3.
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Pellet by centrifugation.
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4.
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Stain for Intracellular Cytokines
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a. Thoroughly resuspend
fixed/permeabilized cells in 50 µl of Perm/Wash™ solution (100 µl
for staining in tubes) containing a pre-determined optimal
concentration of a fluorochrome-conjugated anti-cytokine antibody
or appropriate negative control. Incubate at 4°C for 30 min in
the dark.
b. Wash cells 2 times with 1X Perm/Wash™ solution
(1 ml/wash for staining in tubes) and resuspend
in Staining Buffer prior to flow cytometric analysis.
c. For most cytokines, cells can be left in Stain
Buffer and analyzed the next day. Extended incubation
prior to analysis may result in reduced fluorescent
signals.
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C.
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Alternative Protocol – Activation and
Intracellular Staining of Whole Blood Technical Protocols
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1.
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Dilute whole blood 1:1 volume to volume
(e.g.100 µl:100 µl) with RPMI1640 medium and mix well.
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2.
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Add cell activator or mitogen to diluted blood e.g., 50
ng/ml PMA + 1 µg/ml calcium ionophore A23187 or PMA + 1 µM
ionomycin (final concentration) in the presence of a protein
transport inhibitor such as GolgiPlug™, cat. no. 555029, containing
brefeldin A or GolgiStop™, cat. no. 554724, containing monensin.
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3.
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Vortex briefly to mix. Aliquot 200 µl
into 12 x 75 mm plastic tubes. Incubate for 4-6 hr in 5% CO2 at 37 C.
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4.
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Add 2 ml PharMLyse™ (cat. no. 555899), vortex, incubate
10 min at RT in the dark.
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5.
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Spin 5 min, 500 X g.
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6.
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Aspirate supernatant. Wash 1 X in Staining Buffer. Spin 5 min at
500 x g. Aspirate supernatant.
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7.
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Continue with steps 3-5 under part B of
the General Methods above.
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D.
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Flow Cytometric Analysis
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Set PMT voltage and compensation using
cell surface staining controls. Set quadrant markers based on blocking
controls, isotype controls, or unstained cells.
The frequencies of cytokine-producing
cells present in activated human PBMC culture can vary widely due to
donor variability. Therefore, cryopreserved cells from a single donor
are useful for longitudinal studies.5,6
For proper flow cytometric analysis, cells stained by this method
should be inspected by light microscopy and/or flow light scatter
pattern to confirm that they are well dispersed. In order to make
statistically significant population frequency measurements, sufficiently
large sample sizes should be acquired during flow cytometric analysis.2
Bivariate dot plots or probability contour plots can be generated
upon data reanalysis to display the frequencies of and patterns
by which individual cells co-express certain levels of cell surface
antigen and intracellular cytokine proteins.2
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E.
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Staining Controls
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1.
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Positive Staining Controls
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The TDS for Pharmingen’s
fluorochrome-conjugated anti-cytokine antibodies describe in vitro culture
systems which can induce detectable frequencies of cytokine-producing
cells at specific time-points. Cells stimulated by these methods can
be used as positive controls for experimental systems. Published
reports of immunofluorescent staining and ELISPOT analysis can also
provide useful information regarding different experimental protocols
for generating cytokine-producing cells.1,12,13
Intracellular Cytokine Positive
control cells
To serve as positive controls for
intracellular cytokine staining, PharMingen offers sets of activated
and fixed leukocyte populations which have been screened for cytokine
production.
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Cell Set
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Cat. No.
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Cytokines Measured
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Human
HiCK-1
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555061
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IL-2, IFN- γ, TNF-α
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HiCK-2
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555062
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IL-3, IL-4, IL-10, IL-13, GM-CSF
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HiCK-3
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555063
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IL-1α, IL-1b, IL-6, IL-12, TNF-α
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Mouse
MiCK-1
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554652
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IL-2, IFN- γ, TNF-α
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MiCK-2
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554653
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IL-3, IL-4, IL-10, GM-CSF
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MiCK-3
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554654
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IL-6, IL-12, MCP-1, TNF-α
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Rat
RiCK-2
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555094
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IL-4, IL-10, GM-CSF
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2.
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Negative Staining Controls
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One of the following three controls can
be used to discriminate specific staining from artifactual staining.
Investigators should choose which staining controls best meet their
research needs. Intracellular cytokine staining techniques and the
use of blocking controls are described in detail by C. Prussin and D.
Metcalfe.5
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a.
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Isotype control: Stain with an
isotype-matched control of irrelevant specificity.
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1.
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Resuspend cell pellet in 50 µl of Perm/Wash™
solution (100 µl for staining in tubes) containing
the isotype control antibody at the same concentration
for the anti-cytokine antibody (< 0.5 µg/106
cells).
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2.
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Incubate 15-30 min at 4°C.
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3.
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Wash cells using the aforementioned
procedure for intracellular staining.
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b.
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Ligand blocking control: Pre-block
anti-cytokine antibody with recombinant cytokine
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1.
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Preincubate fluorochrome-conjugated antibodies
with appropriately-diluted cytokine in a volume
≥ 50 µl of Perm/Wash™ solution
at 4°C for 30 min.
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2.
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Resuspend fixed/permeabilized cells
in 50 µl (100 µl for staining in tubes) of pre-blocked labeled
anti-cytokine antibody (in Perm/Wash™ solution) and incubate 30
min at 4°C.
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3.
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Wash cells using the aforementioned
procedure for intracellular staining.
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c.
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Antibody blocking control: Pre-incubate cells with
unconjugated antibody.
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1.
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Resuspend fixed/permeabilized cells in 25 µl
Perm/Wash™ solution (50 µl for staining in
tubes) containing unconjugated anti-cytokine antibody
(same clone as conjugated antibody) diluted to
the appropriate concentration (>5 µg/106
cells), and incubate 30 min at 4°C .
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2.
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After incubation, add fluorochrome
labeled anti-cytokine antibody at an optimal concentration in
25 µl Perm/Wash™ buffer (50 µl for staining in tubes) for a final
volume of 50 µl for staining in microwell plates or 100 µl for
staining in tubes, and incubate 30 min at 4°C.
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3.
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Wash cells using the aforementioned
procedure for intracellular staining.
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SOLUTIONS
Staining Buffer
•Dulbecco’s PBS (DPBS)
•3% heat-inactivated FCS
•0.09% (w/v) sodium azide
•Adjust buffer pH to 7.4 - 7.6, filter (0.2 µm pore membrane), and
store at 4ºC
References:
1.
Carter, L. L., and S.L.
Swain. 1997. Single cell analyses of cytokine
production. Curr. Opin. In Immunology. 9: 177-182.
2.
Parks, D.R., L.A.
Herzenberg, and L.A. Herzenberg. 1989. Flow cytometry and
fuorescence-activated cell sorting. In Fundamental Immunology, 2nd Edition. W.E. Paul, ed. Raven Press
Ltd., New York, p. 781-802.
3.
Jung, T., U. Schauer,
C. Heusser, C. Neumann and C. Rieger. 1993. Detection of
intracellular cytokines by flow cytometery. J. Immunol. Meth. 159:
197-207.
4.
Vikingson, A., K.
Pederson and D. Muller. 1994. Enumeration of IFN-g producing lymphocytes by flow cytometery and correlation with
quantitative measurement of IFN-g. J. Immunol. Meth. 173: 219-228.
5.
Prussin, C. and D.
Metcalfe. 1995. Detection of intracytoplasmic cytokine
using flow cytometry and directly conjugated anti-cytokine
antibodies. J. Immunol. Meth. 188: 117-128.
6.
Elson, L. H., T.B.
Nutman, D. D. Metcalfe and C. Prussin. 1995. Flow cytometric
analysis for cytokine production identifies Th1, Th2, and Th0 cells
within the human CD4+ CD27- lymphocyte subpopulation. J. Immunol. 154:
4294-4301.
7.
Assenmacher, M., J.
Schmitz and A. Radbruch. 1994. Flow cytometric
determination of cytokines in activated murine T helper lymphocytes: Expression of interleukin-10 in
interferon-g and in interleukin-4-expressing cells. Eur. J. Immunol. 24:
1097-1101.
8.
Picker, L. J., M.K.
Singh, Z. Zdraveski, J. R. Treer, S. L. Waldrop, P. R. Bergstresser, and V. C.
Maino. 1995. Direct demonstration of cytokine
synthesis heterogeneity among human memory/effector T cells by flow
cytometry. Blood. 86: 1408-1419.
9.
Sallusto, F., C. R.
Mackay, and A. Lanzavecchia. 1997. Selective expression
of the eotaxin receptor CCR3 by human T helper 2 cells. Science. 277:
2005-2007.
10. Austrup, F., D. Vestweber, E. Borges, M. Lohning, R.
Brauer, U. Herz, H. Renz, R. Hallmann, A. Radbruch, and A. Hamann. 1997. P- and E-selectin
mediate recruitment of T-helper-1 but not T-helper-2 cells into inflamed
tissues. Nature. 385: 81-83.
11. Sander, B., J. Andersson and U. Andersson. 1991. Assessment of cytokines by immunofluorescence and the
paraformaldehyde-saponin procedure. Immunol. Rev. 119: 65-93.
12. Sander, B., I. Hoiden, U. Andersson, E. Moller, and
J. Abrams. 1993. Similar frequencies and kinetics of
cytokine producing cells in murine peripheral blood and spleen. J. Immunol. Meth. 166:
201-214.
13. Andersson, U. and J. Andersson. 1994. Immunolabeling of cytokine producing cells in tissues and
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