Recombinant mouse il 12

Manufactured by BioLegend

Sourced in United States

Recombinant mouse IL-12 is a cytokine that plays a key role in the immune response. It is produced by antigen-presenting cells and stimulates the production of interferon-gamma by T cells and natural killer cells.

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Lab products found in correlation

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Spelling variants of this product

IL-12 (68 citations) Recombinant IL-12 (5 citations) Mouse IL-12 (2 citations)

10 protocols using recombinant mouse il 12

Th1, Th17, and iTreg Cell Induction

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Th1 and Th17 cells were induced as previously described (31 (link)). In brief, 1 ×10⁵ naïve CD4 cells were activated with anti-CD3 (145-2C11, Biolegend) and anti-CD28 (37.51, Biolegend) antibodies in presence of 50 ng/ml mouse recombinant IL-12 or 5 ng/ml of human recombinant TGFβ (Biolegend) and 25 ng/ml mouse recombinant IL-6 (Peprotech) for 3 days. For iTreg induction cells were cultured with 50 ng/ml of human recombinant TGFβ with 100 IU/ml recombinant IL-2 (Chiron), as previously described (32 (link)). For T cell proliferation cells were stained with 5 uM Cell Trace Violet (Thermo Fisher Scientific) in PBS for 15 min at RT, washed with cell culture media, counted and plated as mentioned earlier. Cells were cultured in RPMI 1640 media containing 2.05 mM L-Glutamine, 10% FBS and 1 × penicillin/streptomycin (GE Healthcare). For some experiments MAPK Kinase Inhibitor PD98059 (Sigma Aldrich) was used.

Koliesnik I.O., Kuipers H.F., Medina C.O., Zihsler S., Liu D., Van Belleghem J.D, & Bollyky P.L. (2020). The Heparan Sulfate Mimetic PG545 Modulates T Cell Responses and Prevents Delayed-Type Hypersensitivity. Frontiers in Immunology, 11, 132.

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Isolation and Stimulation of Murine CD4+ T Cell Subsets

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Naïve CD4⁺ T cells were purified by negative selection using a MACS system (Miltenyi Biotec) or isolated by sorting CD4⁺CD25⁻CD44^lowCD62L⁺ T cells by FACS. MP T cells were isolated by sorting CD4⁺CD25⁻CD44^highCD62L⁻ cells. GFP-Egr2⁻ and GFP-Egr2⁺ MP T cells were isolated by sorting GFP-Egr2⁻CD4⁺CD25⁻CD44^highCD62L⁻ and GFP-Egr2⁺CD4⁺CD25⁻CD44^highCD62L⁻ cells, respectively. Purified CD4⁺ T cells were stimulated with plate-bound anti-CD3 at 5 μg/ml (BD Biosciences) and anti-CD28 (2 μg/ml; BD Biosciences) antibodies for 24 h before harvest. MP CD4 T cells were stimulated with 100 ng/ml mouse recombinant IL-12 (BioLegend) for 24 h, or left unstimulated, before analysis of IFNγ-producing cells by intracellular cytokine staining.
For analysis of Egr2, FoxP3, or T-bet expression, the cells were processed using the Foxp3 staining kit (eBioscience). For analysis of cytokine producing cells, the cells were stimulated with 50 ng/ml PMA and 200 ng/ml ionomycin in the presence of Golgistop (BD Biosciences) for 3 h before analysis of cytokine producing cells using the Foxp3 staining kit (eBioscience) and flow cytometry.

Symonds A.L., Zheng W., Miao T., Wang H., Wang T., Kiome R., Hou X., Li S, & Wang P. (2020). Egr2 and 3 control inflammation, but maintain homeostasis, of PD-1high memory phenotype CD4 T cells. Life Science Alliance, 3(9), e202000766.

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Phosphorylated STAT4 and STAT3 Detection

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Following staining for surface markers, cells were incubated in 400 ng ml⁻¹ Recombinant Mouse IL-12 (p70; carrier free; BioLegend; for the detection of phosphorylated STAT4) or Recombinant Mouse IL-6 (carrier free; BioLegend; for the detection of phosphorylated STAT3) for 4 min at room temperature, in a 96-well U-bottom plate. Cells were then centrifuged at 575g for 1 min at room temperature and the supernatant was discarded by flicking. Cells were then incubated in pre-warmed 1× BD Phosflow Lyse/Fix Buffer (BD Biosciences) for 10 min at 37 °C and centrifuged at 1,860g for 2 min. Supernatant was discarded by flicking. Cells were then washed twice with eBioscience Flow Cytometry Staining Buffer (Invitrogen) as described above. Following this, cells were incubated in −20 °C pre-chilled BD Phosflow Perm Buffer III (BD Biosciences) for 30 min on ice. Cells were washed three times in eBioscience Flow Cytometry Staining Buffer (Invitrogen) as above. Finally, cells were stained with either PE-conjugated Mouse anti-Stat4 (pY693; clone 38/p-Stat4) or PE-conjugated Mouse anti-Stat3 (pY705; clone 4/P-STAT3) (both from BD Biosciences), followed by two washes in eBioscience Flow Cytometry Staining Buffer (Invitrogen) as described above. Samples were acquired on a BD LSRFortessa through BD FACSDiva version 8.0 (BD Biosciences).

Ng S.S., De Labastida Rivera F., Yan J., Corvino D., Das I., Zhang P., Kuns R., Chauhan S.B., Hou J., Li X.Y., Frame T.C., McEnroe B.A., Moore E., Na J., Engel J.A., Soon M.S., Singh B., Kueh A.J., Herold M.J., de Oca M.M., Singh S.S., Bunn P.T., Aguilera A.R., Casey M., Braun M., Ghazanfari N., Wani S., Wang Y., Amante F.H., Edwards C.L., Haque A., Dougall W.C., Singh O.P., Baxter A.G., Teng M.W., Loukas A., Daly N.L., Cloonan N., Degli-Esposti M.A., Uzonna J., Heath W.R., Bald T., Tey S.K., Nakamura K., Hill G.R., Kumar R., Sundar S., Smyth M.J, & Engwerda C.R. (2020). The NK cell granule protein NKG7 regulates cytotoxic granule exocytosis and inflammation. Nature immunology, 21(10), 1205-1218.

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Cytokine and Adhesion Molecule Assay

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Recombinant mouse IL-23, E-selectin and P-selectin Fc-chimeras were from R&D Systems (Minneapolis, MN). Recombinant mouse IL-12, IL-2, IL-6, TNF-α, recombinant human TGF-β, and the following antibodies to mouse cytokines and adhesion molecules: IL-4 (clone 11B11), IFNγ (clone XMG 1.2), IL-2 (clone JES6-1A12), CD4 (clone GK 1.5), CD3 (clone145-2C11), CD28 (clone 37.51), IL-17A (clone 2C11-18H10.1), CD43 activation-associated glycoform (clone 1B11), and CD44 (clone IM7) are all from Biolegend (San Diego, CA). Anti- PSGL-1 and mouse TNF-α were purchased from BD-Pharmingen (San Jose, CA), and carrier free CCL20 from Peprotech (Rocky Hill, NJ). PMA and ionomycin were from SIGMA (St. Louis, MO). Secondary Abs coupled to alkaline phosphatase were from Promega (Madison, WI). Vibrant CFSE and Alexa 680 were from Life Technologies (Carlsbad, CA). Myelin Oligodendrocyte glycoprotein was purchased from Anaspec (Fremont, CA) and Pertussis Toxin was purchased from List Biological Laboratories (Campbell, CA). Anti-E-selectin (clone 9A9) antibody was generously provided by Dr. F. William Luscinskas (Brigham and Women's Hospital, Boston, MA) and IgG control was from Biolegend (San Diego, CA).

Velázquez F., Grodecki-Pena A., Knapp A., Salvador A.M., Nevers T., Croce K, & Alcaide P. (2015). CD43 functions as an E-selectin ligand for Th17 cells in vitro and is required for rolling on the vascular endothelium and Th17 cell recruitment during inflammation in vivo. Journal of immunology (Baltimore, Md. : 1950), 196(3), 1305-1316.

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Lung Cancer Cell Line Cultivation

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Human lung cancer cell lines were kind gifts from Professor Pan-Chyr Yang (National Taiwan University, Taipei, Taiwan)^{66 (link)}. The LL2 murine lung cancer cell line was a kind gift from Professor Chao-Liang Wu. The LL2and A549 cells were grown in Dulbecco’s modified Eagle medium (Gibco, Carlsbad, CA, USA). Both culture media were supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 U/ml of penicillin, and 100 mg/ml of streptomycin (Hyclone, Logan, UT, USA). The cells were maintained at 37 °C in a 5% CO₂ incubator. HHT was purchased from Sigma-Aldrich (H0635; St. Louis, MO, USA) and Toronto Research Chemicals (H596500; Toronto, Ontario, Canada). NAR was purchased from Cayman Chemical (20361; Ann Arbor, MI, USA). Both compounds were dissolved in ethanol (2 mM or 50 mg/ml) and diluted in phosphate buffered saline (PBS), 2 µM for in vitro experiments and 2.5 mg/ml for animal experiments. Recombinant mouse IL-12 was purchased from BioLegend (577006; San Diego, CA, USA). Recombinant IL-12 was diluted in PBS (2.5 or 0.25 mg/ml) for animal experiments.

Weng T.Y., Wu H.F., Li C.Y., Hung Y.H., Chang Y.W., Chen Y.L., Hsu H.P., Chen Y.H., Wang C.Y., Chang J.Y, & Lai M.D. (2018). Homoharringtonine induced immune alteration for an Efficient Anti-tumor Response in Mouse Models of Non-small Cell Lung Adenocarcinoma Expressing Kras Mutation. Scientific Reports, 8, 8216.

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IL-33-Induced Treg Suppression of T Cell Proliferation

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Bulk CD4⁺ T cells were purified from PBS- or IL-33-treated, B6 FIR mice and stained for flow sorting (FACSAria, BD Biosciences, San Jose, CA) of RFP⁺(Foxp3⁺) CD4⁺ ST2⁺ or ST2⁻ Treg. Alternatively, splenic B6 FIR CD4⁺ T cells were cultured with IL-33-exposed BALB/c DC and Treg were sorted after 5 d. Purity was consistently greater than 92%. Sorted Treg were tested for their ability to suppress CD3/CD28 T-activator bead (5×10⁴; Life Technologies)-induced, CTV-labeled B6 CD4⁺ or CD8⁺ T cell proliferation at a ratio of 8:1 T effector to Treg (1×10⁵:1.25×10⁴). Where indicated, recombinant mouse IL-12 (5 ng/ml; BioLegend) alone or in combination with IL-33 (10 ng/ml) was used to induce IFN-γ in CD8⁺ T cell cultures. Cultures were harvested on d 4 for flow cytometric analysis of proliferation and IFN-γ by intracellular flow analysis.

Matta B.M., Lott J.M., Mathews L.R., Liu Q., Rosborough B.R., Blazar B.R, & Turnquist H.R. (2014). IL-33 is an unconventional alarmin that stimulates IL-2 secretion by dendritic cells to selectively expand IL-33R/ST2+ regulatory T cells. Journal of immunology (Baltimore, Md. : 1950), 193(8), 4010-4020.

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Murine Model of Inflammatory Bowel Disease

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DSS (molecular weight: 36,000 to 50,000) was purchased from MP Biomedicals (Santa Ana, USA). Dimethyl sulphoxide (DMSO), sulfasalazine (SASP) (purity: 98%), hexadecyltrimethylammonium bromide, hematoxylin, eosin, and o-dianisidine dihydrochloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum, L-glutamine, penicillin, streptomycin, and minimum essential medium were purchased from Invitrogen (Carlsbad, CA, USA). CD4⁺CD62L⁺ T Cell Isolation Kits were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). The fluorescent antibodies CD4, IFN-γ, IL-17A, FoxP₃, and CD3/CD28 were obtained from eBioscience (San Diego, CA, USA) or BD Biosciences (San Jose, CA, USA). Antibodies against p-AMPK, AMPK, Nrf-2, p-STAT1, STAT1, p-STAT3, STAT3, and β-actin were supplied from Cell Signaling Technology Inc. (Beverly, MA, USA). Recombinant mouse IL-12, IL-6, and TGF-β proteins and antibodies IL-4 and IFN-γ were purchased from BioLegend (San Diego, CA, USA). IFN-γ, IL-17A/F, TNF-α, and IL-1β ELISA kits were purchased from eBioscience (San Diego, CA). Total superoxide dismutases (T-SOD), CAT, and GSH-Px colorimetric activity assay kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China). An enhanced chemiluminescence kit was supplied by GE Healthcare Bio-Sciences Corp. (Piscataway, NJ, USA).

Xiao H.T., Peng J., Wen B., Hu D.D., Hu X.P., Shen X.C., Liu Z.G., He Z.D, & Bian Z.X. (2019). Indigo Naturalis Suppresses Colonic Oxidative Stress and Th1/Th17 Responses of DSS-Induced Colitis in Mice. Oxidative Medicine and Cellular Longevity, 2019, 9480945.

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Naive CD4 T Cell Differentiation

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Naïve CD4 T cells were electronically sorted by gating on CD44^lo cells. In case of T helper cell differentiation of Il15^−/− CD4 T cells, additional parameters such as CD62L⁺, CD25⁻, CD1d tetramer⁻ and CD69⁻ were used to define naïve CD4 T cells. Sorted cells were stimulated with plate-bound anti-CD3/CD28 antibodies and cultured under skewing conditions for 5 days. Th1 condition: recombinant mouse IL-12 (10 ng/ml) and anti-IL-4 antibodies (10 µg/ml, Biolegend). Th2 condition: anti-IFNγ antibodies (10 µg/ml), and recombinant mouse IL-4 (20 ng/ml, Peprotech); Th17 conditions: anti-IL-4 (10 µg/ml) and anti-IFNγ antibodies (10 µg/ml), recombinant human TGFβ (5 ng/ml, Peprotech) and recombinant mouse IL-6 (30 ng/ml, Peprotech). Where indicated, recombinant human IL-15 (25 ng/ml, Peprotech), recombinant human IL-2 (25 ng/ml, Peprotech), or recombinant mouse IL-15/IL-15Rα complex (100 ng/ml, eBioscience) was added to the cell cultures. For cytokine production analysis, cultures were stimulated for 3 hours with PMA/ionomycin (Sigma) and Brefeldin A (eBioscience) for 3 hours, followed by intracellular staining and flow cytometric analysis.

Waickman A.T., Ligons D.L., Hwang S., Park J.Y., Lazarevic V., Sato N., Hong C, & Park J.H. (2017). CD4 effector T cell differentiation is controlled by IL-15 that is expressed and presented in trans. Cytokine, 99, 266-274.

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Murine T Cell Polarization Assay

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Anti-mouse CD3e (100340), anti-mouse CD28 (102116), recombinant mouse IL-2 (575406), recombinant mouse IL-6 (575702), recombinant mouse TGF-β (763102), recombinant mouse IL-12 (577004), recombinant mouse IL-23 (589002), Brilliant Violet 421-conjugated anti-mouse CD4 (100443), PerCP anti-mouse CD8a (100731), PE-conjugated anti-mouse/human CD44 (103008), APC-conjugated anti-mouse CD62L (104412), PE/Cyanine7-conjugated anti-mouse IFN-γ (505826), Brilliant Violet 421-conjugated anti-mouse IL-17A (512321), PE-conjugated anti-mouse IL-17A (506904), Alexa Fluor^® 647 anti-mouse/rat/human FOXP3 (320014), FITC anti-mouse CD11c (117306), Brilliant Violet 421™ anti-mouse I-A/I-E (107620), PE/Cy7 anti-mouse CD86 (105014), and PE anti-mouse F4/80 (123110) were purchased from Biolegend (San Diego, CA, USA). Anti-phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199) (4228S), anti-PI3 Kinase p85 (4292S), anti-phospho-AKT (Ser473) (D9E) XP^® Rabbit mAb (4060S), anti-AKT (9272S), anti-PKM2 (4053S) and anti-β-Actin (3700S) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA); anti-PGK1 (A12686), anti-ENO1 (A11448), and anti-LDHA (A1146) antibodies were got from Abclonal (Wuhan, China); anti-HK2 (22029-1-AP) and anti-Glut1 (21829-1-AP) antibodies were obtained from Proteintech (Wuhan, China); and 740 Y-P (HY-P0175) was ordered from Medchem Express (Shanghai, China).

Zou Y., Zhang J., Sun F., Xu Q., Chen L., Luo X., Wang T., Zhou Q., Zhang S., Xiong F., Kong W., Yang P., Yu Q., Liu S, & Wang C.Y. (2024). Fluvoxamine inhibits Th1 and Th17 polarization and function by repressing glycolysis to attenuate autoimmune progression in type 1 diabetes. Molecular Medicine, 30, 23.

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Aβ1-42 Immunization for Alzheimer's Research

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Mice were immunized at 2 m of age by a footpad injection of Aβ1–42 (100 μg; GenScript, Piscataway, NJ) emulsified in CFA H37Ra (Difco, Detroit, MI). The Aβ1–42 peptide used for immunization was initially dissolved in a small volume of DMSO to enhance solubility and then diluted to 2 mg/ml in PBS. The peptide was emulsified with CFA to a final concentration of 1 mg/ml. Ten days later, popliteal, inguinal, and iliac lymph nodes were extracted and the cells were seeded (5 × 10⁶ cells/ml in a 24-well culture dish) in a complete RPMI medium (10% FCS, 10 mM HEPES, 1 mM sodium pyruvate, 10 mM non-essential amino acids, 1% Pen/Strep/Nystatin, and 50 μM 2-ME) supplemented with 20 μg/ml Aβ1–42. Every other day thereafter, human rIL-2 (20 U/ml) in complete RPMI was added to the culture. Following the first week and every 2 w thereafter, T cells (10⁵ T cells/ml) were re-stimulated with irradiated (6000 rad) spleenocytes (5 × 10⁶ cell/ml) in 24-well plates. For Th1 polarization, neutralizing anti–IL-4 (20 μg/ml, clone 11B11; BioLegend, San-Diego, CA) and recombinant mouse IL-12 (1 ng/ml; BioLegend) were added to the culture in the first three stimulations, at seeding, and then 2 d later.

Eremenko E., Mittal K., Berner O., Kamenetsky N., Nemirovsky A., Elyahu Y, & Monsonego A. (2019). BDNF-producing, amyloid β-specific CD4 T cells as targeted drug-delivery vehicles in Alzheimer's disease. EBioMedicine, 43, 424-434.

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