Cytokine Stimulation Protocols

Flow Cytometry Intracellular Staining Quick Guides


Mouse Cytokines: Intracellular Staining Quick Guide
Mouse CytokineCell SourceActivationIncubation TimeRestimulationIntracellular BlockAntibody
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 CytokineCell SourceActivationIncubation TimeRestimulationIntracellular BlockAntibody
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 (Proliferatin 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