Il 12

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Germany, Chile, Switzerland, Canada

IL-12 is a lab equipment product that measures the levels of interleukin-12 (IL-12) in samples. IL-12 is a cytokine that plays a crucial role in the regulation of the immune response. The IL-12 product provides a reliable and accurate way to quantify IL-12 levels, which can be used for research and diagnostic applications.

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Close spelling variants of this product

Mouse IL-12/IL-23 total p40 ELISA Ready-Set-Go Recombinant human IL-12 Human IL-12 p70 ELISA Ready-SET-Go kit Total IL-12 ELISA Kit Mouse IL-12 IL-12/IL-18 ELISA kits for IL-12 p70 Anti-mouse IL-12 IL-12 subunit p40 Recombinant murine IL-12

341 protocols using il 12

Osteoclast Differentiation Modulation

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RAW264.7 (4.5 × 10⁴) cells were seeded into 48-well plate and differentiated into osteoclasts in the presence of the receptor activator of NF-κB ligand (RANKL, 75 ng/mL; Peprotech) with or without proinflammatory cytokines (IL-12, IL-23, IFN-γ, and IL-17 alone: 75 ng/mL; IL-12 + IL-23: 37.5 ng/mL each; IL-12 + IL-23 + IFN-γ + IL-17: 18.75 ng/mL each; Peprotech). The medium was changed every 2 days. After 3 days of osteoclast differentiation, the cells were fixed with 4% PFA and stained with a TRAP kit (Sigma-Aldrich).

Xu J., Li J., Hu Y., Dai K., Gan Y., Zhao J., Huang M, & Zhang X. (2020). IL-23, but not IL-12, plays a critical role in inflammation-mediated bone disorders. Theranostics, 10(9), 3925-3938.

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Cytokine Profiling in Murine EAE Model

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Splenocyte (1 × 10⁶ per well) derived from PF-treated and control EAE mice were cultured in the presence of MOG_35–55 peptide (20 μg/ml) for 48 h. Culture supernatants were collected and the concentrations of IFN-γ (eBioscience, 88–7314), IL-17 (eBioscience, 88–7371), IL-4 (eBioscience, 88–7044), IL-10 (eBioscience, 88–7105), IL-12 (eBioscience, 88–7121), IL-6 (eBioscience, 88–7064), TGF-β (eBioscience, 88–8350), and TNF-α (eBioscience, 88–7324) were measured by ELISA according to the manufacturer’s instruction. For measuring cytokines secreted by BMDCs, BMDCs were stimulated by LPS (100 ng/ml). Supernatants were collected and evaluated for IL-6, IL-12, and IL-23 (eBioscience, 88–7230) by ELISA.

Zhang H., Qi Y., Yuan Y., Cai L., Xu H., Zhang L., Su B, & Nie H. (2017). Paeoniflorin Ameliorates Experimental Autoimmune Encephalomyelitis via Inhibition of Dendritic Cell Function and Th17 Cell Differentiation. Scientific Reports, 7, 41887.

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Polarizing CD4+ T cells in vitro

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Sorted naive CD4⁺ T cells (1 × 10⁵ per well) were cultured with DCs (1 × 10⁵ per well) in the presence of plate-bound anti-CD3 (1 μg ml⁻¹) plus soluble anti-CD28 (2 μg ml⁻¹) and Th polarizing cytokines: IL-12 (4 ng ml⁻¹) for Th1, IL-4 (10 ng ml⁻¹) for Th2, IL-6 (20 ng ml⁻¹) plus TGF-β (5 ng ml⁻¹) for Th17 and TGF-β (5 ng ml⁻¹) for Treg. IL-12 and IL-6 were purchased from eBioscience. After 3 days of culture, cells were collected and analysed for gene expression by qPCR.

Zhao Y., Chu X., Chen J., Wang Y., Gao S., Jiang Y., Zhu X., Tan G., Zhao W., Yi H., Xu H., Ma X., Lu Y., Yi Q, & Wang S. (2016). Dectin-1-activated dendritic cells trigger potent antitumour immunity through the induction of Th9 cells. Nature Communications, 7, 12368.

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Stimulation of NK cell cytokine production

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NK cells were prepared from bone marrow or splenocytes of RAG^−/− mice (B6.12957-RAG1tm1Mom, Jackson). Bone marrow from RAG^−/− mice was cultured in complete media in the presence of recombinant human IL-2 (Proleukin) for seven days. On day 7, NK cells were harvested, plated at 3×10⁵ cells/well and stimulated with 0, 0.1, 0.5, 1, 5 or 10 ng/ml IL-12 (eBioscience). At each concentration of IL-12, NK cells were either stimulated with 50 U/ml TNF-α (eBioscience), 10 ng/ml Flt3L (eBioscience), 100 ng/ml Flt3L, or left unstimulated (37 (link), 38 (link)). Cells were incubated for 48 hours and IFN-γ production was measured by ELISA. Splenocytes from RAG^−/− mice were cultured for 48 hours under the conditions described above, and expression of CD69 and KLRG1 was measured on NK cells by flow cytometry.

Dupont C.D., Pritchard G.H., Hidano S., Christian D.A., Wagage S., Muallem G., Tait Wojno E.D, & Hunter C.A. (2015). Flt3L is essential for survival and protective immune responses during toxoplasmosis. Journal of immunology (Baltimore, Md. : 1950), 195(9), 4369-4377.

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Transcription Factor and Cytokine Modulation in Splenocytes

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Following extracellular staining, cells were fixed with FoxP3/Transcription Factor Staining Buffer Set for 30 min at room temperature and washed with 1x Permeabilization buffer (eBiosciences). Transcription factor antibodies were diluted in 1x FoxP3 Permeabilization buffer and cells were incubated for 30 min at room temperature.
To assess Eomes downregulation after culture in IL-12, 5 × 10⁶ splenocytes were cultured in R-10 medium (RPMI-1640 medium containing 10% FBS, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin) with 300 IU/mL IL-2 with 20 ng/mL IL-12 (Pepro Tech). Unstimulated control wells contained only 300 IU/mL IL-2.
For detection of IFNγ production, 5 × 10⁶ splenocytes were cultured for 5 hr in R-10, with the addition of 20 ng/mL IL-12, 20 ng/mL IL-12 + 5 ng/mL IL-18, or 0.5 μg/mL PMA + 4 μg/mL Ionomycin. Brefeldin A was added after 1 hr.

Park E., Patel S., Wang Q., Andhey P., Zaitsev K., Porter S., Hershey M., Bern M., Plougastel-Douglas B., Collins P., Colonna M., Murphy K.M., Oltz E., Artyomov M., Sibley L.D, & Yokoyama W.M. (2019). Toxoplasma gondii infection drives conversion of NK cells into ILC1-like cells. eLife, 8, e47605.

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Differentiation of Human Naive CD4+ T Cells

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Human naïve CD4⁺ T cells were activated and differentiated under certain conditions. Briefly, cells were cultured under the stimulation of precoated anti-CD3 antibody (2 μg/ml, Sigma-Aldrich, USA) and anti-CD28 antibody (1 μg/ml, Sigma-Aldrich) for the desired time with the intention of activation. In addition to anti-CD3 and anti-CD28 antibodies, human naïve CD4⁺ T cells were cultured under the following polarization conditions for cell differentiation: anti-IL-4 antibody (10 μg/ml, Peprotech, USA), IL-12 (10 ng/ml, Peprotech), IL-2 (5 ng/ml, Peprotech) for Th1 polarization; anti-IFN-γ antibody (10 μg/ml, Peprotech), IL-2 (5 ng/ml), IL-4 (25 ng/ml, Peprotech) for Th2 polarization; anti-IFN-γ antibody (10 μg/ml), anti-IL-4 antibody (10 μg/ml), IL-6 (25 ng/ml, Peprotech), TGF-β (5 ng/ml, R&D System, USA), IL-1β (12.5 ng/ml, Peprotech), IL-21 (20 ng/ml, R&D System), IL-23 (25 ng/ml, Peprotech) for Th17 polarization; IL-6 (20 ng/ml), IL-21 (20 ng/ml), IL-12 (10 ng/ml), TGF-β (5 ng/ml) for Tfh polarization, IL-2 (10 ng/ml), TGF-β (5 ng/ml) for Treg polarization.

Lu S., Wei X., Zhu H., Hu Z., Zheng M., Wu J., Zhao C., Yang S., Feng D., Jia S., Zhao H, & Zhao M. (2023). m6A methyltransferase METTL3 programs CD4+ T-cell activation and effector T-cell differentiation in systemic lupus erythematosus. Molecular Medicine, 29, 46.

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Cytokine-induced CD4+ T cell activation

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Naïve CD4⁺ T cells (1 ×10⁵ cell/well) from HC subjects (n = 6) were stimulated with dynabeads^® human T-activator CD3/CD28 (Invitrogen, USA) in flat-bottom 96-well plates, and cultured in the presence of IL-12 (PeproTech, USA) (10 ng/ml), IL-21 (PeproTech, USA) (20 ng/ml), IL-12 + IL-21, or IL-17 (PeproTech, USA) (10 ng/ml) for 72 hours, respectively.

Du B., Teng J., Yin R., Tian Y., Jiang T., Du Y, & Cai W. (2021). Increased Circulating T Follicular Helper Cells Induced via IL-12/21 in Patients With Acute on Chronic Hepatitis B Liver Failure. Frontiers in Immunology, 12, 641362.

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Tolerance Induction in Human MDMs

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For tolerance induction in vitro, human MDMs (0.5×10⁶) were pretreated with 100 μg/ml MDP (Bachem, King of Prussia, PA) for 48h prior to extensive wash and retreated for 24h with 100 μg/ml MDP. In some cases anti-IL-10 or anti-TGFβ neutralizing antibodies (R&D Systems, Minneapolis, MN) or lipid A (Peptide International, Louisville, KY) was used. Supernatants were assayed for cytokine secretion per manufacturer instructions using the following antibodies: TNF, IL-6, IL-8, IL-10 (BD Biosciences, San Jose, CA) or IL-12 (eBioscience, San Diego, CA).

Zheng S., Hedl M, & Abraham C. (2015). Twist1 and Twist2 contribute to cytokine downregulation following chronic NOD2 stimulation of human macrophages through the coordinated regulation of transcriptional repressors and activators. Journal of immunology (Baltimore, Md. : 1950), 195(1), 217-226.

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Cytokine-mediated PFC activation assay

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PFCs were resuspended in complete RPMI-1640 medium, stimulated with 2 μg/mL peptides plus 1 μg/mL anti-CD28, 1 μg/mL anti-CD49d mAbs, or PMA (20 ng/ml; Sigma Aldrich, Saint Louis, MO, USA) plus ionomycin (1 μg/mL; Sigma-Aldrich). PFCs were cultured in the presence of medium alone, IL-6 (30 ng/mL; Peprotech), IL-12 (5 ng/ml; eBioscience, Santiago, Chile), IL-21 (50 ng/mL; Peprotech), IL-27 (40 ng/ml; eBioscience) alone, E/C peptides, or E/C peptides plus cytokines.

Li L., Jiang Y., Lao S., Yang B., Yu S., Zhang Y, & Wu C. (2016). Mycobacterium tuberculosis-Specific IL-21+IFN-γ+CD4+ T Cells Are Regulated by IL-12. PLoS ONE, 11(1), e0147356.

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Human MDMs Cytokine Response to Stimuli

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Human MDMs were treated with lipid A (Peptides International, Louisville, KY), Pam3Cys (EMD Millipore, Billerica, MA), polyinosinic-polycytidylic acid [poly(I:C)], CpG DNA (Invivogen, San Diego, CA), IL-12, IFN-γ (R&D Systems, Minneapolis, MN), IFN-α (Biovision, Milpitas, CA), or cocultured with Salmonella enterica serovar Typhimurium at multiplicity of infection (MOI) 10:1. Supernatants were assayed for IL-6, IL-10, TNF, IL-1β, IFN-γ (BD Biosciences), IL-12 (eBioscience, San Diego, CA), or IFN-γ (BioLegend) by ELISA. In some cases, cells were pretreated with 5 μg/ml fludarabine (STAT1 inhibitor), 150 μM lisofylline (STAT4 inhibitor) (Cayman Chemical Company), Upadacitinib (JAK1 inhibitor), or BMS-986165 (TYK2 inhibitor) (MedChemExpress).

Hedl M., Sun R, & Abraham C. (2020). Disease Risk-Associated Genetic Variants in STAT1 and STAT4 Function in a Complementary Manner to Increase Pattern-Recognition Receptor–Induced Outcomes in Human Macrophages. Journal of immunology (Baltimore, Md. : 1950), 205(5), 1406-1418.

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