Introduction
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
| IL-1aNEW! | mouse PEC | mINFg (100ng/ml)(2h4)/LPS (100ng/ml)(22hr) | 2hr/22hr | - | Monensin | ALF-161 |
| IL-1bNEW! | mouse PEC | mINFg (100ng/ml)(2h4)/LPS (100ng/ml)(22hr) | 2hr/22hr | - | Monensin | B122 |
| IL-2 | mouse spleen | ConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4 (20ng/ml) (3d) | 2d/3d | anti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hr | Monensin | JES6-5H4 |
| IL-4 | mouse spleen | ConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4 (20ng/ml) (3d) | 2d/3d | anti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hr | Monensin | BVD6-24G2 |
| IL-6 | mouse spleen | ConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4 (20ng/ml) (3d) | 2d/3d | anti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hr | Monensin | MP5-20F3 |
| IL-10 | mouse spleen | ConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4 (20ng/ml) (3d) | 2d/3d | anti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hr | Monensin | JES5-16E3 |
| IL-12 | mouse PEC | mIFNg (100ng/ml) (2hr)/LPS (100ng/ml) (22hr) | 2hr/22hr | - | Monensin | C17.8 |
| GM-CSF | mouse spleen | ConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4 (20ng/ml) (3d) | 2d/3d | anti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hr | Monensin | MP1-22E9 |
| IFN-g | mouse spleen | ConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4 (20ng/ml) (3d) | 2d/3d | anti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hr | Monensin | XMG1.2 |
| TNF-a | mouse spleen | ConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4 (20ng/ml) (3d) | 2d/3d | anti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hr | Monensin | MP6-XT22 |
| TNF-aNEW! | mouse spleen | ConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4 (20ng/ml) (3d) | 2d/3d | anti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hr | Monensin | TN3-19.12 |
| 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 |
Table 2: Human Cytokine Intracellular Staining Quick Guide
| 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) |
Intracellular Cytokine Staining Protocol Precautionary Note Prior to using antibodies, do a quick spin to recover the whole volume in small vials. 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. For optimal performance of fluorochrome conjugated antibodies, wrap the vials with aluminum foil and store at 4°C in the dark. Do not freeze. 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 Prepare target cells of interest (see specific instructions). 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 . 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. Add 1 ml of Permeabilization Buffer to each tube, centrifuge for 5 minutes and aspirate supernatant. Repeat step 4. Resuspend cells in 100 µl of Permeabilization Buffer and incubate in the dark at room temperature for 5 minutes. 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. Incubate in the dark at room temperature for 20 minutes. Add 1 ml of Permeabilization Buffer, centrifuge for 5 minutes and aspirate supernatant. Resuspend the cell pellet in 0.5 ml of Flow Staining Buffer. Run on a flow cytometer and analyze. Table 3: Mouse Phenotyping Quick Guide
| 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 |
