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Intracellular Cytokine Staining Protocol

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A modification of the basic immunofluorescence staining and flow cytometric analysis protocol can be used for the simultaneous analysis of surface molecules and intracellular cytokines at single-cell level. In this protocol, cells are first activated in vitro, stained for surface antigens, as in the regular staining protocol, then fixed and permeabilized to allow for anti-cytokine antibodies to stain intracellularly. In vitro stimulation of cells is usually required for detection of cytokines by flow cytometry since cytokine levels are typically too low in freshly-prepared cells. Stimulation of cells with the appropriate reagent will depend on the cell type and the experimental conditions. For example, to stimulate T cells to produce IFN-gamma, TNF-alpha, IL-2, and IL-4, a combination of PMA (a PKC activator) and Ionomycin (a calcium ionophore) or anti-CD3 antibodies are used. To induce IL-6, IL-10 or TNF-alpha production by monocytes, stimulation of PBMC's with LPS is used.

Note: Optimal stimulation period for induction of a given cytokine is variable and has to be determined. For example, the best time for detection of IL-6-producing cells by human LPS-activated monocytes is 6 hours, whereas IL-10 needs at least 24 hours stimulation.

In contrast to detection of secreted cytokines by ELISA, for detection of intracellular cytokines, it is necessary to block secretion of cytokines with reagents such as Monensin or Brefeldin A during the last few hours of the stimulation. It is advised that the investigators evaluate the use and efficacy of different protein transport inhibitors in their specific assay system.

Note: Generally, the non-specific background staining of fixed and permeabilized cells is higher than surface staining; therefore, extra protein such as BSA or FCS can be added to the staining buffer. Optimal concentration of the fluorochrome-conjugated anti-cytokine antibodies has to be determined experimentally. To confirm specificity of the staining, it is common to block the directly-conjugated anti-cytokine antibodies with excess amounts of unlabeled antibody. Alternatively, recombinant cytokines can be used for blocking.

eBioscience antibodies are available as Functional Grade (FG) Purified (sterile, endotoxin-tested, no azide), Purified, FITC, Cy5, PE, PE-Cy5, and APC conjugates. For more information about the properties of fluorescent dyes, please visit our Fluorescent Dyes page in our BestProtocols. The Functional Grade Purified format is recommended for neutralization studies. More antibodies are in development; if you do not see an antibody of your choice, please send us an e-mail so we may provide you timely product updates.

Table 1: Mouse Cytokine Intracellular Staining Quick Guide
Mouse CytokineCell SourceActivationIncubation TimeRestimulationIntracellular BlockAntibody
IL-1aNEW!mouse PECmINFg (100ng/ml)(2h4)/LPS (100ng/ml)(22hr)2hr/22hr-MonensinALF-161
IL-1bNEW!mouse PECmINFg (100ng/ml)(2h4)/LPS (100ng/ml)(22hr)2hr/22hr-MonensinB122
IL-2mouse spleenConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4 (20ng/ml) (3d)2d/3danti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hrMonensinJES6-5H4
IL-4mouse spleenConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4 (20ng/ml) (3d)2d/3danti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hrMonensinBVD6-24G2
IL-6mouse spleenConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4 (20ng/ml) (3d)2d/3danti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hrMonensinMP5-20F3
IL-10mouse spleenConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4 (20ng/ml) (3d)2d/3danti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hrMonensinJES5-16E3
IL-12mouse PECmIFNg (100ng/ml) (2hr)/LPS (100ng/ml) (22hr)2hr/22hr-MonensinC17.8
GM-CSFmouse spleenConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4 (20ng/ml) (3d)2d/3danti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hrMonensinMP1-22E9
IFN-gmouse spleenConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4 (20ng/ml) (3d)2d/3danti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hrMonensinXMG1.2
TNF-amouse spleenConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4 (20ng/ml) (3d)2d/3danti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hrMonensinMP6-XT22
TNF-aNEW!mouse spleenConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4 (20ng/ml) (3d)2d/3danti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hrMonensinTN3-19.12
Annotations: mouse PEC=mouse thioglycolate-elicited peritoneal macrophages; ConA=Concanavalin A; Iono=Ionomycin; LPS=Lipopolysaccharide; PMA=Phorbol Myristate Acetate; 2d=2 day culture; 5hr=5 hour culture

Human Cytokine Cell Source Activation Incubation Time Restimulation Intracellular Block Antibody
IL-1aNEW! PBMC LPS 100ng/ml 24hr - Monensin CRM8
IL-1bNEW! PBMC LPS 100ng/ml 24hr - Monensin CRM56
IL-2 PBMC PMA (30-50ng/ml)/Iono (1ug/ml) 5hr - Monensin MQ1-17H12
IL-4 PBMC anti-CD3 (10µg/ml, immobilized) + anti-CD28 (2µg/ml, soluble) + IL-2 (10ng/ml) + IL-4 (20ng/ml) (2d); IL-2 (10ng/ml) + IL-4 (20ng/ml) (3d) 2d/3d PMA (5ng/ml) + Ionomycin (500ng/ml) (4hr) Monensin MP4-25D2
IL-6 PBMC LPS 100ng/ml 5hr - Monensin MQ2-13A5
IL-10 PBMC LPS 100ng/ml 24hr - Monensin JES3-9D7
IL-12 PBMC hIFNg (100ng/ml) (2hr)/LPS (100ng/ml) (22hr) 2hr/22hr - Monensin C8.6
IFN-g PBMC PMA (30-50ng/ml)/Iono (1ug/ml) 5hr - Monensin 4S.B3
TNF-a PBMC PMA (30-50ng/ml)/Iono (1ug/ml) 5hr - Monensin MAb11
Annotations: Iono=Ionomycin; PMA=Phorbol Myristate Acetate; LPS=Lipopolysaccharide; 2d=2 day culture; 5hr=5 hour culture; LPS for activation of human PBMC obtained from Sigma (#L-8274)

Precautionary Note

  1. Prior to using antibodies, do a quick spin to recover the whole volume in small vials.
  2. We monitor our conjugated antibodies to make sure they are void of aggregation. Some protocols include a step of airfuging the antibody prior to multi-color flow analysis.
  3. For optimal performance of fluorochrome conjugated antibodies, wrap the vials with aluminum foil and store at 4°C in the dark. Do not freeze.
  4. When staining mouse antigens please note that fixation and/or delayed analysis may yield poor signal-to-noise for certain antigens. For best results, stain fresh mouse cells.

What You Need

Materials

Directly fluorochrome-conjugated antibodies (see charts above) or Th1/Th2 Flow Panel

Cells to be stained

12 x 75 mm round-bottom test tubes (Falcon Cat. No. 2008) or 96-well round-bottom microtiter plates

Buffers

eBioscience IC Fixation Buffer, Cat. No. 00-8222

eBioscience Permeabilization Buffer, Cat. No. 00-8333

Instruments

Pipettes and pipettors

Centrifuge

Ice bucket or refrigerator

Flow Cytometer

Experiment Duration

Stimulation (variable depending on the cytokine of interest)

2-3 hours staining

Method

  1. Prepare target cells of interest (see specific instructions).
  2. Stain cell-surface antigen following the Surface Staining Protocol. The choice of the surface marker depends on the experimental question. For suggestions on best phenotypic markers for different cell types please see the appropriate BestPhenotyping Markers Chart: Mouse or Human.
  3. After the last wash, fix the cells by adding 100 µl of Fixation Solution while vortexing the tube and incubate in the dark at room temperature for 20 minutes.
  4. Add 1 ml of Permeabilization Buffer to each tube, centrifuge for 5 minutes and aspirate supernatant.
  5. Repeat step 4.
  6. Resuspend cells in 100 µl of Permeabilization Buffer and incubate in the dark at room temperature for 5 minutes.
  7. Dilute the optimal concentration of fluorochrome-conjugated anti-cytokine mAb in 20 µl Permeabilization Buffer and add to the appropriate tube. Mix by vortexing. A good range of conjugated antibody for this application is 0.5-0.06 µg/106 cells. eBioscience fluorochrome-conjugated anti-cytokine antibodies are also available in 20 µl/test sizes. If using these reagents, add 20 µl of pre-titrated antibody to the appropriate tube.
  8. Incubate in the dark at room temperature for 20 minutes.
  9. Add 1 ml of Permeabilization Buffer, centrifuge for 5 minutes and aspirate supernatant.
  10. Resuspend the cell pellet in 0.5 ml of Flow Staining Buffer.
  11. Run on a flow cytometer and analyze.

 
Cell Type Antigen Antibody Order Info Comments
T Lymphocytes CD3 145-2C11 14-0031 pan, developmentally regulated
T Subsets:



T cytotoxic, CD8+ CD8 53-6.7 14-0081 mature and developing T cells
T helper, CD4+ CD4 RM4-5 14-0042 mature and developing T cells


GK1.5 14-0041
T regulatory, CD4+CD25+ CD25 PC61.5 14-0251
Natural T, NK+ NK1.1 PK136 14-5941 small subset CD3+DX5+ or CD3+NK1.1+

CD49b/DX5 DX5 14-5971
B Lymphocytes CD19 MB19-1 14-0191 pre- to mature B, not on plasma


6D5 14-0192

CD45R/B220 RA3-6B2 14-0452 also on NK progenitors, LAK, activated T
Natural Killer Cells (NK) CD49b/DX5 DX5 14-5971 on all strains tested

NK1.1 PK136 14-5941 only on NK1.1+ strains

CD94 18d3 14-0941
Dendritic Cells (DC) CD11c N418 14-0114 also on some T cells

DEC-205 NLDC-145 n/a dim

33D1 antigen 33D1 14-5884 splenic DC
Monocytes/Macrophages CD11b M1/70 14-0112 also on some T and NK cells

F4/80 CI:A3-1 n/a not on blood monocytes

CD115/M-CSFR AFS98 14-1152

CD14 Sa2-8 14-0141 not on blood monocytes
Granulocytes Ly6G, GR-1 RB6-8C5 14-5931 blood neutrophils, BM granulocytes and myeloid lineage
Erythrocytes TER-119 TER-119 14-5921 early to mature red cells, not on CFU-E
Endothelial Cells CD31 390 14-0311 also low level on leukocytes and platelets

CD106/VCAM 429 14-1061 upregulated on cytokine activated endothelium, also on BM myeloid cells

MECA-32 MECA-32 n/a
Platelets CD41 MWReg30 n/a also on megakaryocytes

CD61 2C9.G3 14-0611 also on activated lymphocytes, granulocytes
Mast Cells IgeRIa high affinity chain MAR-1 14-5898

 
Cell Type Antigen Antibody Order Info Comments
T Lymphocytes CD3 HIT3a 14-0039 pan, developmentally regulated


OKT3 14-0037 pan, developmentally regulated


UCHT1 14-0038 pan, developmentally regulated

CD2 RPA-2.10 14-0029 immature and mature T

CD5 UCHT2 14-0059 immature and mature T

CD6 M-T605 n/a immature and mature T

CD7 coming soon

T Subsets:



T cytotoxic, CD8+ CD8 RPA-T8 14-0088 mature and developing T cells
T helper, CD4+ CD4 RPA-T4 14-0049 mature and developing T cells
T regulatory, CD4+CD25+ CD25 BC96 14-0259
Natural T, NK+ CD56 MEM188 14-0569 small subset, CD3+CD56+
B Lymphocytes CD19 HIB19 14-0199 Pre- to mature B, not on plasma

CD20 2H7 14-0209 Pre- to mature B, not on plasma
Natural Killer Cells (NK) CD56 MEM188 14-0569 virtually all NK

CD94 DX22 14-0949 NK subset, T subset

CD158a HP-3E4 n/a NK subset, T subset
Dendritic Cells (DC) CD83 HB15e 14-0839 also on some germinal center and cell lines

CD205/DEC-205 MG38 14-2059

CD197/CCR7 CCR7.6B3 14-9977

CD209/DC-SIGN eB-h209 14-2099
Monocytes CD14 61D3 14-0149
Endothelial Cells CD31 WM59 14-0319 also low level on leukocytes and platelets

CD106 STA 14-0419 upregulated on cytokine activated endothelium
Platelets CD62P/P-Selectin AK-4 14-0628 in cytoplasmic stores of endothelium and platelets, transported to surface on activation
Mast Cells IgeRIa high affinity chain AER-37 (CRA-1) 14-5899
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