Intracellular cytokine and chemokine detection often requires stimulation of defined cell types prior to analysis. Refer to this list below to learn how to induce commonly analyzed cytokine and chemokine expression, as well as the antibody clones we offer to detect each.

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
Cytokine/Chemokine Clone(s) Target Cells Stimulation Stimulation period Protein transport blocker Surface marker
IL-1α 364-3B3-14 PBMCs LPS (100 ng/ml) 4-6 hours monensin or brefeldin A CD14*
IL-1β JK1B-1 PBMCs LPS (100 ng/ml) 4-6 hours monensin or brefeldin A CD14*
IL-2 MQ1-17H12 PBMCs PMA (50 ng/ml) plus ionomycin (1 μg/ml) 4-6 hours monensin or brefeldin A CD3
IL-3 BVD3-1F9 Purified CD4+ T cells stimulated with immobilized anti-CD3 (10 μg/ml) + soluble anti-CD28 (5 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 (50 ng/ml) plus ionomycin (1 μg/ml) 4-6 hours monensin or brefeldin A CD4*
IL-4 MP4-25D2 or 8D4-8 PBMCs PMA (50 ng/ml) plus ionomycin (1 μg/ml) 4-6 hours monensin or brefeldin A CD4*
IL-5 JES1-39D10 Purified CD4+ T cells stimulated with immobilized anti-CD3 (10 μg/ml) + soluble anti-CD28 (5 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 (50 ng/ml) plus ionomycin (1 μg/ml) 4-6 hours monensin or brefeldin A CD4*
IL-6 MQ2-13A5 PBMCs LPS (100 ng/ml) 4-6 hours monensin or brefeldin A CD14*
IL-8 JK8-1 PBMCs LPS (100 ng/ml) 4-6 hours monensin or brefeldin A CD14*
IL-9 MH9A4 Purified CD4+ T cells stimulated with immobilized anti-CD3 (10 μg/ml) + soluble anti-CD28 (5 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 (50 ng/ml) plus ionomycin (1 μg/ml) 4-6 hours monensin or brefeldin A CD4*
IL-10 JES3-9D7 or JES3-19F1 PBMCs LPS (100 ng/ml) 18-24 hours monensin or brefeldin A CD14*
IL-12 p40 C11.5 PBMCs IFN-γ (100 ng/ml) prime for 2 hours, then add LPS (1 μg/ml) 18-24 hours monensin or brefeldin A CD14*
IL-13 JES10-5A2 Purified CD4+ T cells stimulated with immobilized anti-CD3 (10 μg/ml) + soluble anti-CD28 (5 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 (50 ng/ml) plus ionomycin (1 μg/ml) 4-6 hours monensin or brefeldin A CD4*
IL-17A BL168 PBMCs PMA (50 ng/ml) plus ionomycin (1 μg/ml) 4-6 hours monensin or brefeldin A CD4*
IL-21 3A3-N2 or 4BG1 PBMCS PMA (50 ng/ml) plus ionomycin (1 μg/ml) 4-6 hours monensin or brefeldin A CD4*
IL-22 BG/IL22 PBMCs PMA (50 ng/ml) plus ionomycin (1 μg/ml) 4-6 hours monensin or brefeldin A CD4*
GM-CSF BVD2-21C11 PBMCs stimulated with soluble anti-CD3 (UCHT1, 1 μg/ml) + soluble anti-CD28 (5 μg/ml) for 2 days, wash and then culture in IL-2 (20 ng/ml) + IL-4 (50 ng/ml) for 3-4 days PMA (50 ng/ml) plus ionomycin (1 μg/ml) 4-6 hours monensin or brefeldin A CD3
IFN-γ 4S.B3 or B27 PBMCs PMA (50 ng/ml) plus ionomycin (1 μg/ml) 4-6 hours monensin or brefeldin A CD3
MCP-1 5D3-F7 or 2H5 PBMCs LPS (100 ng/ml) 18-24 hours monensin or brefeldin A CD14*
TNF-α MAB11 PBMCs PMA (50 ng/ml) plus ionomycin (1 μg/ml) 4-6 hours monensin or brefeldin A CD3
TNF-β 359-81-11 PBMCs stimulated with soluble anti-CD3 (UCHT1, 1 μg/ml) + soluble anti-CD28 (5 μg/ml) for 2 days, wash and then culture in IL-2 (20 ng/ml) + IL-4 (50 ng/ml) for 3-4 days PMA (50 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.

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