See below for intracellular staining quick guides for mouse and human cytokines as well as for non-cytokine proteins.
See step-by-step Intracellular Staining Protocols
Mouse cytokines: intracellular staining quick guide for flow cytometry
| Mouse cytokine | Cell source | Activation | Incubation time | Restimulation | Intracellular block | Antibody |
|---|---|---|---|---|---|---|
| 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 | Brefeldin A | MP1-22E9 |
| IFN-gamma | 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 | Brefeldin A | XMG1.2 |
| IL-1 alpha | mouse PEC | mIFNγ (100ng/mL) (2hr)/LPS (100ng/mL)(22hr) | 2hr/22hr | - | Brefeldin A | ALF-161 |
| IL-1 beta | mouse PEC | LPS (100 ng/mL) (22hr) | 22hr | - | Monensin | NJTEN3 |
| 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 | Brefeldin A | JES6-5H4 |
| IL-4 | mouse spleen | Th2 polarized | 6d | PMA (50 ng/mL) + Ionomycin (1 ug/mL) 5hr | Brefeldin A | BVD6-24G2, 11B11 |
| IL-5 | mouse splenic CD4 | ConA (3ug/mL) (2d)/IL-2 (20ng/mL)+IL-4 (20ng/mL) (3d) | 2d/3d | immobilized) + anti-CD28 (2ug/mL soluble) 5hr | Brefeldin A | TRFK5 |
| IL-6 | mouse PEC | LPS (100 ng/mL) (22hr) | 22hr | - | 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 | Brefeldin A | JES5-16E3, JES5-2A5 |
| IL-12/IL-23 (p40) | mouse PEC | LPS (100 ng/mL) (22hr) | 22hr | - | Brefeldin A | C17.8 |
| IL-13 | mouse spleen | Th2 polarized | 6d | PMA (50 ng/mL) + Ionomycin (1 ug/mL) 5hr | Brefeldin A | eBio13A |
| IL-17A | mouse spleen | Th17 polarized | 6d | PMA (50 ng/mL) + Ionomycin (1 ug/mL) 5hr | Monensin | eBio17B7 |
| IL-17F | mouse spleen | Th17 polarized | 6d | PMA (50 ng/mL) + Ionomycin (1 ug/mL) 5hr | Monensin | eBio18F10 |
| IL-21 | mouse spleen | Th17 polarized | 9d | PMA (50 ng/mL) + Ionomycin (1 ug/mL) 5hr | Monensin | FFA21 |
| IL-22 | mouse spleen | Th17 polarized | 12d | PMA (50 ng/mL) + Ionomycin (1 ug/mL) 5hr | Brefeldin A | IL22JOP |
| IL-23 p19 | mouse bone marrow | mGM-CSF (40 ng/mL) | 8d | LPS (1 ug/mL) (24 hr) | Monensin | fc23cpg |
| MCP-1/ CCL2 | mouse PEC | LPS (100 ng/mL) (24hr) | 24hr | - | Brefeldin A | 2H5 |
| TNF alpha | 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 | Brefeldin A | MP6-XT22, TN3-19 |
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 cytokines: intracellular staining quick guide
| Human cytokine | Cell source | Activation | Incubation time | Restimulation | Intracellular block | Antibody |
|---|---|---|---|---|---|---|
| G-CSF | PBMC | LPS (1 ug/mL) | 24hr | - | Monensin | 8F5CSF |
| GM-CSF | PBMC | PMA (30-50ng/mL)/Iono (1ug/mL) | 5hr | - | Monensin | BVD2-21C11 |
| IFN gamma | PBMC | PMA (30-50ng/mL)/Iono (1ug/mL) | 5hr | - | Brefeldin A | 4S.B3 |
| IL-1 alpha | PBMC | LPS (1 ug/mL) | 24hr | - | Monensin | 364/3B3-14, CRM8 |
| IL-1 beta | PBMC | LPS (100ng/mL) | 4hr | - | Brefeldin A | CRM56 |
| IL-1RA | PBMC | LPS (100ng/mL) | 24hr | - | Brefeldin A | CRM17 |
| IL-2 | PBMC | PMA (30-50ng/mL)/Iono (1ug/mL) | 4-6hr | - | Brefeldin A | MQ1-17H12 |
| IL-4 | PBMC | PMA (30-50ng/mL)/Iono (1ug/mL) | 4-6hr | - | Brefeldin A | 8D4-8 |
| IL-5 | CD4 | Th2 polarizing cultures | 6d | PMA (50ng/mL) + Ionomycin (1ug/mL) (5hr) | Brefeldin A | TRFK5, JES1-5A10 |
| IL-6 | PBMC | LPS (100ng/mL) | 24hr | - | Brefeldin A | MQ2-13A5 |
| IL-9 | CD4 | Th2 polarizing cultures | 6d | PMA (50ng/mL) + Ionomycin (1ug/mL) (5hr) | Monensin | MH9A4 |
| IL-10 | CD4 | Th2 polarizing cultures | 6d | PMA (50ng/ml) + Ionomycin (1ug/ml) (5hr) | Monensin | JES3-9D7 |
| IL-12/ IL-23 (p40) | PBMC | hIFN gamma (100ng/mL) (2hr)/LPS (100ng/mL) (22hr) | 2hr/22hr | - | Brefeldin A | C8.6 |
| IL-13 | CD4 | anti-CD3 (10ug/mL, immobilized) + anti-CD28 (2ug/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) | Brefeldin A | PVM13-1 |
| IL-17A | PBMC | Th17 polarizing cultures | 6d | PMA (50ng/ml) + Ionomycin (1ug/ml) (5hr) | Brefeldin A | eBio64CAP17, eBio64DEC17 |
| IL-21 | PBMC | PMA (30-50ng/mL)/Iono (1ug/mL) | 4-7hr or 12-18hr | - | Brefeldin A | eBio3A3-N2 |
| IL-22 | CD4 | Th17 polarizing cultures | 6d | PMA (50ng/ml) + Ionomycin (1ug/ml) (5hr) | Brefeldin A | IL22JOP |
| IL-23 p19 | PBMC | hGM-CSF (40ng/mL) +hIL-4 (40ng/mL) | 6d | LPS (1ug/mL) (24hr) | Monensin | 23dcdp |
| MCP-1/ CCL2 | PBMC | LPS (1ug/mL) | 24hr | - | Monensin | 2H5, 5D3-F7 |
| RANTES/ CCL5 | PBMC | LPS (1ug/mL) | 24hr | - | Monensin | VL1 |
| TNF alpha | PBMC | PMA (30-50ng/mL)/Iono (1ug/mL) | 5hr | - | Brefeldin A | MAb11 |
| TNF beta | PBMC | Th1 polarizing cultures | 6d | PMA (50ng/mL) + Ionomycin (1ug/mL) (5hr) | Monensin | 359-81-11 |
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)
Non-cytokine proteins: intracellular staining quick guide
| Antigen | Antibody |
|---|---|
| Mouse/Rat Bcl-2 | 10C4 |
| Mouse CTLA-4 (CD152) | UC10-4B9 |
| Human CTLA-4 (CD152) | 14D3 |
| Human/Mouse/Rat Cytochrome C | 6H2 |
| Human Foxp3 | PCH101 |
| Mouse Foxp3 | FJK-16s |
| GATA3 | |
| Mouse/Human Granzyme B | eBioGrB |
| House Langerin (CD207) | eBioRMUL.2 |
| Human Nanog | hNanog.1 |
| Human PCNA (Proliferating Cell Nuclear Antigen) | PC10 |
| Mouse Perforin | eBioOMAK-D |
| Human Perforin | dG9 |
| ROR gamma | |
| Mouse SLP-76 | MS76 |
| Human SLP-76 | HS76 |
| Human/Mouse T-bet | eBio4B10 (4B10, 4-B10) |
| Human TLR3 | TLR3.7 |
| Mouse TLR9 | M9.D6 |
| Human TLR9 | eB72-1665 |
| Mouse/Human ZAP-70 | 1E7.2 |
For Research Use Only. Not for use in diagnostic procedures.