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Recombinant human il 12

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Recombinant human IL-12 is a cytokine that plays a critical role in the regulation of the immune system. It is produced by antigen-presenting cells and promotes the differentiation of naive T cells into Th1 cells, enhancing cell-mediated immunity.

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Lab products found in correlation

Il 18, by R&D Systems (4 mentions) Il 1β, by Thermo Fisher Scientific (3 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (3 mentions) Il 23, by Thermo Fisher Scientific (3 mentions) Ficoll paque plus, by GE Healthcare (3 mentions) Il 21, by Thermo Fisher Scientific (2 mentions) Il 23, by R&D Systems (2 mentions) Efluor660 anti il 21, by Thermo Fisher Scientific (2 mentions) Anti cd45ra fitc, by Thermo Fisher Scientific (2 mentions) Anti cd4 apc cy7, by BD (2 mentions) Anti icos pe, by Thermo Fisher Scientific (2 mentions) Anti cd25 pe cy7, by BD (2 mentions) Recombinant human il 15, by R&D Systems (2 mentions) Cd4 t cell isolation kit, by STEMCELL (2 mentions) 96 well round bottom plate, by Corning (2 mentions) Anti human cd3 ucht1, by BD (2 mentions) Soluble anti human cd28 28.2, by BD (2 mentions) Sgk1 inhibitor gsk650394, by Bio-Techne (2 mentions) Akt inhibitor mk2206, by Bio-Techne (2 mentions) Frizzled 8 fc chimera protein, by R&D Systems (2 mentions)

Close spelling variants of this product

IL-12 (213 citations) Recombinant IL-12 (7 citations) Human IL-12 (3 citations) BCG plus recombinant human IL-12 (2 citations)

27 protocols using recombinant human il 12

1

Multiparameter Immunophenotyping of T Cells

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Alexa488-conjugated anti-GATA3, Alexa647-conjugated anti-CXCR5, APC-Cy7-conjugated anti-CD4, BUV395-conjugated anti-IFNγ, BV711-conjugated anti-IL-2, Pe-Cy7-conjugated anti-CD25, PE-conjugated anti-CCR6 and anti-mouse IgG1, and PerCpCy5.5-conjugated anti-CD127 and anti-Tbet were purchased from Becton Dickinson. Alexa488-conjugated anti-IL-10, eFluor660-conjugated anti-IL-21, FITC-conjugated anti-CD45RA, PE-conjugated IL-22, Pe-Cy7-conjugated anti-IL-4 and mouse IgG1 were purchased from eBiosciences. APC-Cy7-conjugated anti-IL-17A, BV421-conjugated anti-CXCR3, and BV605-conjugated anti-TNFα was purchased from Biolegend. FITC-conjugated anti-CCR7 and recombinant human IL-12 was purchased from R&D Systems. Anti-DOCK8 mAb was purchased from Santa Cruz Biotechnology. Recombinant human TGFβ, IL-1β, IL-6, IL-21 and IL-23 were from Peprotech. Prostaglandin E2, PMA, calcium ionophore (ionomycin), Brefeldin A, and saponin were purchased from Sigma-Aldrich and recombinant human IL-4 was provided by Dr Rene de Waal Malefyt (DNAX Research Institute, Palo Alto, CA). T cell activation and expansion (TAE) beads (anti-CD2/CD3/CD28) were purchased from Miltenyi Biotec and CFSE was purchased from Invitrogen.
Tangye S.G., Pillay B., Randall K.L., Avery D.T., Phan T.G., Gray P., Ziegler J.B., Smart J.M., Peake J., Arkwright P.D., Hambleton S., Orange J., Goodnow C.C., Uzel G., Casanova J.L., Reyes S.O., Freeman A.F., Su H.C, & Ma C.S. (2016). DOCK8-DEFICIENT CD4+ T CELLS ARE BIASED TO A TH2 EFFECTOR FATE AT THE EXPENSE OF TH1 AND TH17 CELLS. The Journal of allergy and clinical immunology, 139(3), 933-949.
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2

Bovine CD4+ T Cell Activation Assay

Cited 3 times
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The culture was similar as described previously [30 (link)]. Briefly, anti-bovine CD3 (Clone #MM1A, WSU, Pullman, WA, USA) was added to 48-well plates at 10 µg/mL in 250 µL 1X PBS (Hyclone, Logan, UT, USA), as in previous reports [30 (link),81 (link),82 (link)]. Anti-CD28 antibody (Clone#CC219, BioRad) was supplemented in culture medium at 5 ug/ml as reported [50 (link)]. Recombinant human IL-12 (R & D Systems, Twin City, MN, USA) was supplemented at a final concentration of 20 ng/mL, as reported by [31 (link),32 (link)]. Sorted, bovine CD4+ T cells were seeded at 1 × 105 per well in 48-well plates, and autologous neutrophils (from the same cattle) were added to the wells of T cells at 10:1 (1 × 106 neutrophils) ratios as determined in our recent report [30 (link)]. Neutralizing Ab against bovine IL-10 (Clone#CC320, BioRad, Hercules, CA, USA) was added to the medium at 10 µg/mL [30 (link),82 (link),83 (link)]. Plates were incubated at 37 °C in an atmosphere of 5% CO2 for 3.5 days and then analyzed for CD25 and CD62L expression in CD4+ T cells. For IFNγ intracellular staining, a fraction of cultured cells was washed and resuspended in Allos medium supplied with cell activation cocktail (R&Dsystems, Minneapolis, MN, USA), incubated for an additional 4 hours before intracellular staining. All stained samples were analyzed using flow cytometry.
Xiao Z., Kandel A, & Li L. (2021). Synergistic Activation of Bovine CD4+ T Cells by Neutrophils and IL-12. Pathogens, 10(6), 694.
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3

Cytokine-Stimulated CD107a Degranulation

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Where stated, cells were stimulated with combinations of recombinant human IL-12 (R&D Systems, Minneapolis, MN, USA), IL-15 (R&D Systems), and IL-18 (R&D Systems) for 12 h at concentrations of 0.5 ng/mL, 20 ng/mL, and 100 ng/mL [24 (link),25 (link),29 (link),30 (link)], respectively, and protein transport inhibitors containing brefeldin A (BD Biosciences, Franklin Lakes, WI, USA) and monensin (BD Biosciences) were added for the last 4 h. For analyzing CD107a degranulation, cells were stimulated with K562 cells (ATCC, Manassas, VA, USA) at an effector to target ratio of 1:1 and anti-CD107a was added during the last 6 h.
Kupke P., Adenugba A., Schemmerer M., Bitterer F., Schlitt H.J., Geissler E.K., Wenzel J.J, & Werner J.M. (2023). Immunomodulation of Natural Killer Cell Function by Ribavirin Involves TYK-2 Activation and Subsequent Increased IFN-γ Secretion in the Context of In Vitro Hepatitis E Virus Infection. Cells, 12(3), 453.
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4

Comprehensive Immunophenotyping Panel

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The following were purchased from eBiosciences: anti-IL-21 eFluor660, anti-IL-10 Alexa Fluor 488, anti-IFNγ BV605, anti-CD45RA FITC, anti-ICOS PE, anti-PD-1 biotin. The following were purchased from Becton Dickinson: anti-CXCR5 Alexa Fluor 647, anti-CD4 APC-Cy7, anti-IL-2 BV711, anti-CD25 PE-Cy7, anti-CD127 PerCP-Cy5.5, SA-PerCpCy5.5, anti-IL-17F BV786, anti-TNFα BUV395, anti-IL-9 PerCP-Cy5.5, anti-IL-13 BV421. The following were purchased from Biolegend: anti-IL-22 PE, anti-IL-4 PE-Cy7, anti-IL-17A APC-Cy7. The TCR Vß repertoire kit was from Beckman Coulter. Recombinant human IL-12 was purchased from R&D Systems.
Bier J., Rao G., Payne K., Brigden H., French E., Pelham S.J., Lau A., Lenthall H., Edwards E.S., Smart J.M., Cole T.S., Choo S., Joshi A.Y., Abraham R.S., O’Sullivan M., Boztug K., Meyts I., Gray P.E., Berglund L.J., Hsu P., Wong M., Holland S.M., Notarangelo L.D., Uzel G., Ma C.S., Brink R., Tangye S.G, & Deenick E.K. (2019). Activating mutations in PIK3CD and murine CD4+ T cells. The Journal of allergy and clinical immunology, 144(1), 236-253.
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5

In vitro Effector T Cell Generation

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To generate in vitro effectors, lymph nodes and spleens of donor TCR transgenic mice (OTII or P25) were harvested and homogenized through a 70μm cell strainer. Following red blood cell lysis, cells underwent CD4 negative T cell selection using magnetic beads (Miltenyi Biotec). Three to 4×106 cells were plated with 1×107 mitomycin C-treated (Sigma Aldrich) C57BL/6 splenocytes, in RPMI 1640 (Lonza) supplemented with 10% non heat-inactivated FCS (Atlanta Biologicals), L-Glutamine, Penicillin-Streptomycin (Lonza), non-essential amino acids (Gibco), and 50μM 2-mercaptoethanol in each well of a 24-well plate. The splenocytes were pulsed with 2μM OVA323-339 peptide (OTII) or 1.26μM Ag85b 240-254 peptide (P25). The cells were cultured in the presence of 10ng/ml IFNγ (eBioscience), 10μg/ml anti-mouse IL-4 (eBioscience), and 10ng/ml recombinant human IL-12 (R&D). On day 2, 10ng/ml recombinant human IL-2 (R&D) was also added to the culture medium. On day 6, cells were harvested and for FACS experiments 2-4×106 cells were transferred into recipient mice, whereas for imaging experiments, about 6-8×106 T cells were transferred. Transferred populations were either congenically marked, genetically labeled with GFP, or labeled with intracellular dyes (CMTMR, CMFDA, TAMRA, or CMF 2HR, all from Invitrogen) for further identification by flow cytometry or imaging.
Torabi-Parizi P., Vrisekoop N., Kastenmuller W., Gerner M.Y., Egen J.G, & Germain R.N. (2014). Pathogen-related differences in the abundance of presented antigen are reflected in CD4+ T cell dynamic behavior and effector function in the lung. Journal of immunology (Baltimore, Md. : 1950), 192(4), 1651-1660.
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6

IFN-γ-Secreting CD4+ T Cell Isolation

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IFN-γ-secreting CD4+ T cells were isolated by flow cytometry following activation with recombinant human IL-12 (500 pg/ml, R&D Systems, Minneapolis, MN), IL-18 (50 ng/ml, R&D Systems) and TL1A (100 ng/ml, Fitzgerald Industries International, Acton, MA) for 8h. IFN-γ-secreting cells were detected using an IFN-γ secretion assay cell enrichment and detection kit (Miltenyi Biotec, San Diego, CA) and sorted on a FACS Aria II (BD Biosciences, San Jose, CA).
Intracellular IFN-γ production and analysis of cellular aggregation was conducted essentially as described.15 (link) Cells were either rested or stimulated for 24h with IL12/IL18 and TL1A and Brefeldin A (10ug/ml) was added for the last 4h. Cells were fixed and stained for intracellular IFN-γ (brilliant violet 421-IFN-γ, eBioscience) or isotype control. Samples were washed and stained for cellular aggregation (propidium iodide). Cells were acquired on a LSRII Flowcytometer (BD Biosciences, San Jose) and analyzed with FlowJo software (TreeStar Inc., Ashland, OR). For LFA1 blocking analysis cells were pre-incubated overnight with monoclonal control mouse IgG1k (15ug/ml) or anti-LFA1 (TS1/18) followed by stimulation with IL12/IL18 and TL1A for 24h.
Gonsky R., Fleshner P., Deem R.L., Biener-Ramanujan E., Li D., Potdar A.A., Bilsborough J., Yang S., McGovern D.P, & Targan S.R. (2017). Association of RNASET2 Gene Polymorphisms with Decreased Expression and Clinical Characteristics of Severity in Crohn’s Disease. Gastroenterology, 153(1), 219-232.
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7

In vitro Differentiation of Human CD4+ T Cell Subsets

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Differentiation of human CD4+ T cell subsets in vitro was performed as previously described (69 (link)–72 (link, link, link)). In brief, naive CD4+ CD45RA+ T cells were enriched from donor PBMCs using the T cell enrichment kit (Miltenyi). Cell culture was in the presence of anti-CD3/CD28 activation beads (1:20 bead to cell ratio) (Invitrogen) plus polarization condition medium (RPMI-1640 containing 10% knockout serum replacement [ThermoFisher]) and supplemented with cytokines (vide infra) at 37 °C for 6 d. For Th1 differentiation conditions, naive T cells were cultured in the presence of anti–IL-4 neutralizing antibody (10 mg/mL; R&D) and recombinant human IL-12 (5 ng/mL; R&D). For Th2 conditions, naive T cells were cultured in the presence of anti–IFN-γ neutralizing antibody (10 mg/mL; R&D) in addition to recombinant IL-4 (4 ng/mL; Peprotech). To obtain differentiated Th17 cells, naive T cells were cultured using IL-1β (20 ng/mL; Peprotech), IL-6 (20 ng/mL; Peprotech), recombinant TGF-β (3 ng/mL; Peprotech), and IL-23 (10 ng/mL; R & D) for 7 d. To expand Th17 cells, Dynabeads (Invitrogen) isolated CD4+ T cells were used instead of CD45RA+ selected T cells.
Cribbs A.P., Terlecki-Zaniewicz S., Philpott M., Baardman J., Ahern D., Lindow M., Obad S., Oerum H., Sampey B., Mander P.K., Penn H., Wordsworth P., Bowness P., de Winther M., Prinjha R.K., Feldmann M, & Oppermann U. (2020). Histone H3K27me3 demethylases regulate human Th17 cell development and effector functions by impacting on metabolism. Proceedings of the National Academy of Sciences of the United States of America, 117(11), 6056-6066.
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8

Effect of IL-12 on Human Retinal Endothelial Cells

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Human retinal microvascular endothelial cells (HRECs; Cell Systems Corporation, Kirkland, WA, USA) were maintained with CS-C complete medium (Cell Systems Corporation) in 6% CO2 at 37 °C. Cells at passages 5–7 were used for the studies.
HRECs suspensions (2 × 104 cells/500 μL) were seeded in 24 wells plates, cultured in C-SC medium with 10% FBS, and allowed the attachment. After starvation in C-SC medium with 1% FBS for 24 h, the medium was replaced with or without recombinant human IL-12 (rIL-12) at 0.1 ng, 1 ng, or 10 ng/mL (R&D System) for 24 h. Then the cells were collected and examined for the mRNA expressions by real-time RT-PCR.
Zhou Y., Yoshida S., Kubo Y., Kobayashi Y., Nakama T., Yamaguchi M., Ishikawa K., Nakao S., Ikeda Y., Ishibashi T, & Sonoda K.H. (2016). Interleukin-12 inhibits pathological neovascularization in mouse model of oxygen-induced retinopathy. Scientific Reports, 6, 28140.
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9

Cytokine-Induced Signaling Pathway Analysis

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Cells were stimulated for 25 min in the presence or absence of 25 ng/ml recombinant human IL-12 (R&D Systems), 50 ng/ml IL-23 (eBioscience) or 25 ng/ml IL-1β (R&D Systems). Cells were then washed two times in FACS buffer and fixed for 20 min in BD Fix and Perm buffer at room temperature (BD Biosciences), washed in FACS buffer, and fixed in 90% ice-cold methanol for 30 min on ice. Cells were washed two times with BD Perm/Wash and stained with antibodies against intracellular markers, including pSTAT4 (Y693) and pSTAT3 (pY705) (both from BD Biosciences) or Ikbα (Cell Signaling), in BD Perm/Wash for 40 min at room temperature. Cells were then washed two times, resuspended in BD Perm/Wash and analyzed by flow cytometry.
Duhen T, & Campbell D.J. (2014). IL-1β promotes the differentiation of polyfunctional human CCR6+CXCR3+ Th1/17 cells that are specific for pathogenic and commensal microbes. Journal of immunology (Baltimore, Md. : 1950), 193(1), 120-129.
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10

Stimulation of Jurkat Cells with miRNA

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Purified Jurkat cells expressing miRσ, miRμ or miRNeg were stimulated in cRPMI containing either 1 μg/mL phytohemagglutinin (PHA alone), PHA and 1 ng/mL Recombinant human IL12 (PHA+IL12Lo), or PHA and 10 ng/mL Recombinant human IL12 (PHA+IL12Hi). Recombinant human IL12 was purchased from R&D Systems (Minneapolis, MN). Two days after stimulation, cells and their supernatants were collected and separated; the cells were counted and lysed for RT-PCR analysis, while the supernatants were frozen and later used for ELISA analysis.
Reeme A.E., Claeys T.A., Aggarwal P., Turner A.J., Routes J.M., Broeckel U, & Robinson R.T. (2018). Human IL12RB1 expression is allele-biased and produces a novel IL12 response regulator. Genes and immunity, 20(3), 181-197.
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