Cytokine/Chemokine Clone(s) Target Cells Stimulation Stimulation period Protein transport blocker Surface marker
IL-1α ALF-161 Thioglycollate-elicited peritoneal macrophage LPS (1 μg/ml) 4-6 hours monensin or brefeldin A CD107b (MAC-3)
IL-2 JES6-5H4 Splenocytes PMA (20 ng/ml) plus ionomycin (1μg/ml) 4-6 hours monensin or brefeldin A CD3
IL-3 MP2-8F8 Purified CD4+ T cells stimulated with immobilized anti-CD3 (10 μg/ml) + soluble anti-CD28 (2 μg/ml) + IL-2 (20 ng/ml) + IL-4 (50 ng/ml) for 2 days, wash and then culture in IL-2 + IL-4 for 3-4 days PMA (20 ng/ml) plus ionomycin (1 μg/ml) 4-6 hours monensin or brefeldin A CD4*
IL-4 11B11 Purified CD4+ T cells stimulated with immobilized anti-CD3 (10 μg/ml) + soluble anti-CD28 (2 μg/ml) + IL-2 (20 ng/ml) + IL-4 (50 ng/ml) for 2 days, wash and then culture in IL-2 + IL-4 for 3-4 day PMA (20 ng/ml) plus ionomycin (1 μg/ml) 4-6 hours monensin or brefeldin A CD4*
IL-5 TRFK Purified CD4+ T cells stimulated with immobilized anti-CD3 (10 μg/ml) + soluble anti-CD28 (2 μg/ml) + IL-2 (20 ng/ml) + IL-4 (50 ng/ml) for 2 days, wash and then culture in IL-2 + IL-4 for 3-4 day PMA (20 ng/ml) plus ionomycin (1 μg/ml) 4-6 hours monensin or brefeldin A CD4*
IL-6 MP5-20F3 Thioglycollate-elicited peritoneal macrophage LPS (1 μg/ml) 4-6 hours monensin or brefeldin A CD107b (MAC-3)
IL-9 RM9A4 Purified CD4+ T cells stimulated with immobilized anti-CD3 (5 μg/ml) + soluble anti-CD28 (5 μg/ml) + IL-2 (20 ng/ml) + IL-4 (10 ng/ml) + TGF-β1 (10 ng/mL) + anti-mouse IFN-γ (10 μg/ mL) for 3 days PMA (500 ng/ml) plus ionomycin (500 ng/ml) 4-6 hours Brefeldin A CD4*
IL-10 JES5-16E3 Purified CD4+ T cells stimulated with immobilized anti-CD3 (10 μg/ml) + soluble anti-CD28 (2 μg/ml) + IL-2 (20 ng/ml) + IL-4 (50 ng/ml) for 2 days, wash and then culture in IL-2 + IL-4 for 3-4 day PMA (20 ng/ml) plus ionomycin (1 μg/ml) 4-6 hours monensin or brefeldin A CD4*
IL-12 C15.6 Thioglycollate-elicited peritoneal macrophage IFN-γ (10 ng/ml) prime for 2 hours, then add LPS (1 μg/ml) 18-24 hours monensin or brefeldin A CD107b (MAC-3)
IL-17A TC11- 18H10.1 Purified CD4+ T cells stimulated with immobilized anti-CD3 (2 μg/ml) + soluble anti-CD28 (5 μg/ml), IL-6 (50 ng/ mL), TGF-β1 (1ng/mL), anti-mouse IL-4 (10 μg/mL), anti-mouse IFN-γ (10 μg/ mL), and 300 nM of FICZ cultured for 3 days. Alternatively, mouse IL-23 (5 ng/ ml) can be used in place of FICZ. PMA (500 ng/ml) + ionomycin (500 ng/ml) 4-6 hours Brefeldin A CD4*
IL-17F 9D3.1C8 Purified CD4+ T cells stimulated with immobilized anti-CD3 (2 μg/ml) + soluble anti-CD28 (5 μg/ml), IL-6 (50 ng/ mL), TGF-β1 (1ng/mL), anti-mouse IL-4 (10 μg/mL), anti-mouse IFN-γ (10 μg/ mL), and 300 nM of FICZ cultured for 3 days. Alternatively, mouse IL-23 (5 ng/ ml) can be used in place of FICZ. PMA (500 ng/ml) + ionomycin (500 ng/ml) 4-6 hours Brefeldin A CD4*
IL-21 BL25168 Purified CD4+ T cells stimulated with immobilized anti-CD3 (2 μg/ml) + soluble anti-CD28 (5 μg/ml), IL-6 (50 ng/ mL), TGF-β1 (1ng/mL), anti-mouse IL-4 (10 μg/mL), anti-mouse IFN-γ (10 μg/ mL), and 300 nM of FICZ cultured for 3 days. Alternatively, mouse IL-23 (5 ng/ ml) can be used in place of FICZ. PMA (500 ng/ml) + ionomycin (500 ng/ml) 4-6 hours Brefeldin A CD4*
IL-22 Poly5164 Purified CD4+ T cells stimulated with immobilized anti-CD3 (2 μg/ml) + soluble anti-CD28 (5 μg/ml), IL-6 (50 ng/ mL), TGF-β1 (1ng/mL), anti-mouse IL-4 (10 μg/mL), anti-mouse IFN-γ (10 μg/ mL), and 300 nM of FICZ cultured for 3 days. Alternatively, mouse IL-23 (5 ng/ ml) can be used in place of FICZ. PMA (500 ng/ml) + ionomycin (500 ng/ml) 4-6 hours Brefeldin A CD4*
IL-27 MM27-7B1 Thioglycolate-elicited Balb/c peritoneal macrophages primed with IFN-γ (100 ng/ml) for 2 hours LPS (1 μg/ml) 4-6 hours monensin or brefeldin A CD107b (MAC-3)
GM-CSF MP1-22E9 Splenocytes stimulated with immobilized anti-CD3 (10 μg/ml) + soluble anti-CD28 (2 μg/ml) for 2 days, wash and then culture in IL-2 (20 ng/ml) + IL-4 (50 ng/ml) for 3-4 day PMA (20 ng/ml) plus ionomycin (1 μg/ml) 4-6 hours monensin or brefeldin A CD3
IFN-γ XMG1.2 Splenocytes PMA (20 ng/ml) plus ionomycin (1 μg/ml) 4-6 hours monensin or brefeldin A CD3
MCP-1 2H5 Thioglycollate-elicited peritoneal macrophage LPS (1 μg/ml) 4-6 hours monensin or brefeldin A CD107b (MAC-3)
TNF-α MP6-XT22 Splenocytes PMA (20 ng/ml) plus ionomycin (1 μg/ml) 4-6 hours monensin or brefeldin A CD3

Notes: *Surface expression of CD4 is down-modulated by PMA/Ionomycin stimulation, and surface expression of CD14 is down- regulated by LPS stimulation.