Mouse intracellular cytokine detection stimulation protocol

Mouse cytokine Cell source Activation-polarization culture Training time Stimulate again Intracellular blocker 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 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 IL-1 alpha Mouse PEC mIFNγ (100 ng/mL) (2 hr) / LPS (100 ng / mL) (22 hr) 2hr/22hr - Brefeldin A IL-1 beta Mouse PEC LPS (100 ng/mL) (22hr) 22hr - Monensin 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 IL-4 Mouse spleen Th2 polarized 6d PMA (50 ng/mL) + Ionomycin (1 ug/mL) 5hr Brefeldin A 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 IL-6 Mouse PEC LPS (100 ng/mL) (22hr) 22hr - Monensin 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 IL-12/IL-23 (p40) Mouse PEC LPS (100 ng/mL) (22hr) 22hr - Brefeldin A IL-13 Mouse spleen Th2 polarized 6d PMA (50 ng/mL) + Ionomycin (1 ug/mL) 5hr Brefeldin A IL-17A Mouse spleen Th17 polarized 6d PMA (50 ng/mL) + Ionomycin (1 ug/mL) 5hr Monensin IL-17F Mouse spleen Th17 polarized 6d PMA (50 ng/mL) + Ionomycin (1 ug/mL) 5hr Monensin IL-21 Mouse spleen Th17 polarized 9d PMA (50 ng/mL) + Ionomycin (1 ug/mL) 5hr Monensin IL-22 Mouse spleen Th17 polarized 12d PMA (50 ng/mL) + Ionomycin (1 ug/mL) 5hr Brefeldin A IL-23 p19 Mouse bone marrow mGM-CSF (40 ng/mL) 8d LPS (1 ug/mL) (24 hr) Monensin MCP-1/ CCL2 Mouse PEC LPS (100 ng/mL) (24hr) 24hr - Brefeldin A 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 Description: mouse PEC=mouse thioglycolate-elicited peritoneal macrophages peritoneal macrophages; ConA=Concanavalin A; Iono=Ionomycin; LPS=Lipopolysaccharide; PMA=Phorbol Myristate Acetate; 2d=2 day Culture; 5hr=5 hour culture; CpG ODN 2395 = unmethylated oligodeoxynucleotide containing CpG structure . Source: http://us.ebioscience.com/resources/best-protocols/flow-cytometry/flow-cytometry-intracellular-staining-quick-guide.htm

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