Intracellular Staining Quick Guides | Thermo Fisher Scientific

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

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