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Rhil 12

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RhIL-12 is a recombinant human interleukin-12 protein. It is a cytokine that plays a role in the regulation of immune responses.

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Lab products found in correlation

Rhil 4, by Thermo Fisher Scientific (3 mentions) Rhil 2, by Thermo Fisher Scientific (3 mentions) Rhil 15, by Thermo Fisher Scientific (2 mentions) Rhil 18, by R&D Systems (2 mentions) Human ab serum, by Merck Group (1 mentions) Golgistop protein transporter inhibitor containing monensin, by BD (1 mentions) Rhil 7, by R&D Systems (1 mentions) Rhil 6, by R&D Systems (1 mentions) Rhil 21, by R&D Systems (1 mentions) Rhil 10, by R&D Systems (1 mentions) Ifn α, by R&D Systems (1 mentions) Zombie aqua, by BioLegend (1 mentions) Intracellular staining buffer kit, by Thermo Fisher Scientific (1 mentions) Tnf α, by BioLegend (1 mentions) Dead cell removal kit, by Miltenyi Biotec (1 mentions) Ifn γ, by BioLegend (1 mentions) Pma ionomycin, by BioLegend (1 mentions) Rhil 18, by MBL Life Science (1 mentions) Anti ifn γ, by Thermo Fisher Scientific (1 mentions) Il 1β, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

RhIL-12 p70 (2 citations)

11 protocols using rhil 12

1

Standardized Cytokine Preparation and Dilution

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SKA-IL-4 and SKA-IL-12 were prepared by GUNA Laboratories (GUNA S.p.a, Milan, Italy) using a standardized method. Cytokines, sequentially diluted in saline solution (serial dilution 1:100), underwent a shaking process (vertical shaking; 10-cm motion range; shaking speed 100 oscillations over 10 seconds, kinetically energized by a mechanically applied force) [18] (link). The preparation was supplied at a concentration of 10− 8 μg/ml. rhIL-4 and rhIL-12 were from PeproTech Inc. (Rocky Hill, NJ).
Radice E., Bellone G, & Miranda V. (2015). Enhancement of the Immunostimulatory Functions of Ex Vivo–Generated Dendritic Cells from Early-Stage Colon Cancer Patients by Consecutive Exposure to Low Doses of Sequential-Kinetic-Activated IL-4 and IL-12. A Preliminary Study. Translational Oncology, 8(4), 327-338.
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2

Cytokine-Induced Memory-Like NK Cells

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For CIML generation, MNCs (3 × 106/ml) were incubated with rhIL-12 (10 ng/ml, Preprotech, Rocky Hill, USA) plus rhIL-15 (1 ng/ml), IL-18 (50 ng/ml) for 16 hours as pre-activated group. MNCs were incubated with rhIL-15 (1 ng/ml) for 16hrs as control group. Control and pre-activated cells were wash 3 times, and cultured in RPMI-1640 with 10% human AB serum (Sigma-Aldrich) plus rhIL-15 (1 ng/ml) for 15 days. Every 2–3 days, half of the medium was discarded and replenished by fresh medium with IL-15 (1 ng/ml)35 (link). Cultured cells were then harvested and stimulated with IL-12 (10 ng/ml), IL-18 (50 ng/ml) plus IL-15 (100 ng/ml) in the presence of GolgiStop protein transporter inhibitor containing monensin (BD Bioscience) for 4 hrs.
Lin S.J., Hsu C.Y., Kuo M.L., Lee P.T., Hsiao H.S, & Chen J.Y. (2020). Phenotypic and functional characterization of natural killer cells in rheumatoid arthritis-regulation with interleukin-15. Scientific Reports, 10, 5858.
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3

Modulation of Human CD8+ T Cell Activation

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Purified naive and total human CD8+ T cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% L-glutamine, respectively. Cells were seeded at 5 to 10×104 cells into 96-well U bottom plate and stimulated with anti-CD3/CD28 coated microbeads (Dynabeads® T-Activator CD3/CD28; Thermo Fisher Scientific, Waltham, MA, USA) in the absence or presence of the indicated cytokines; recombinant human (rh) IL-1β (25 ng/ml), rhIL-6 (6.25 to 100 ng/ml), rhIL-21 (0.1 to 100 ng/ml), rhIL-7 (50 ng/ml), IFN-α (50 or 100 ng/ml), rhIL-10 (6.25 to 100 ng/ml; all from R&D systems, Minneapolis, MN, USA), rhIL-12 (50 ng/ml), rhIL-15 (50 or 100 ng/ml), rhIL-2 (100 IU/ml, all from PeproTech, Rocky Hill, NJ, USA). In some experiments, cells were stimulated with anti-CD3/CD28 coated microbeads with chemical inhibitors: STAT1 inhibitor Fludarabin, STAT3 inhibitor 5,15-DPP, or STAT5 inhibitor CAS 285986-31-4 (all from Sigma-aldrich, St. Louis, MO, USA).
Kim D.H., Kim H.Y, & Lee W.W. (2021). Induction of Unique STAT Heterodimers by IL-21 Provokes IL-1RI Expression on CD8+ T Cells, Resulting in Enhanced IL-1β Dependent Effector Function. Immune Network, 21(5), e33.
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4

Isolation and Polarization of ILC2 Cells

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Week 6 H1-CAR ILC2 were purified from mechanically dissociated ATOs using the Dead Cell Removal Kit (Miltenyi, Auburn CA), followed by staining with PE-anti-CD8 and anti-PE MicroBeads (Miltenyi, Auburn CA) to deplete CD8+ T and NK/ILC1 cells. 1.5×105 ILC2-enriched cells were plated in 96-well U-bottom plates as per proliferation assays, above. For type 1 polarization, 20 ng/mL rhIL-12 (Peprotech) was added in addition to rhIL-7 and rhIL-2. For type 2 polarization, rhIL-25, rhIL-33, and rhTSLP were added in addition to rhIL-7 and rhIL-2 at 20 ng/ml each. On day 5, PMA/ionomycin with protein transport inhibitor cocktail (eBioscience, San Diego, CA) was added to each well for 6 hours. Cells were washed and stained for surface markers and Zombie Aqua (Biolegend, San Diego, CA) prior to fixation and permeabilization with an intracellular staining buffer kit (eBioscience, San Diego, CA) and intracellular staining with antibodies against IFNγ, TNFα, IL-5, and IL-13 (Biolegend, San Diego, CA).
Weinreich M., Liang C, & Stillman B. (1999). The Cdc6p nucleotide-binding motif is required for loading Mcm proteins onto chromatin. Proceedings of the National Academy of Sciences of the United States of America, 96(2), 441-446.
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5

Peripheral Blood Mononuclear Cell Activation Protocol

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PBMCs from healthy adult peripheral blood samples were isolated by Ficoll centrifugation. Human PBMCs were plated at 3–5 × 106 cells/mL and preactivated overnight (about 16 h) using rhIL-12 (10 ng/mL, PeproTech) + rhIL-18 (50 ng/mL, MBL International) + rhIL-15 (1 ng/mL, PeproTech) or control (rhIL-15, 1 ng/mL) conditions, washed three times to remove cytokines, and then added with rhIL-15 (1 ng/mL) to maintain the culture in a complete RPMI 1640 medium containing 10% fetal bovine serum. After a week of culture at 37°C in a 5% CO2 incubator, the cells were washed, added with PMA + Ionomycin (cell activation cocktail with brefeldin was obtained from BioLegend (San Diego, CA), the same set of cytokines IL-12/15/18, K562 tumor cell lines, 0.1–1 µM of the CDK4/6 inhibitor, Myc inhibitor, DNA-demethylating agent, and enhancer of zeste homolog2 (EZH2) inhibitor separately for 5 h), and then washed three times with phosphate-buffered saline.
Zhu S., Zhang C., Sun Q., Wang Y., Yu W., Wei F, & Ren X. (2022). Trained Immunity of IL-12-, IL-15-, and IL-18-Induced CD3+CD56+ NKT-Like Cells. Journal of Oncology, 2022, 8724933.
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6

Isolation and Differentiation of CD4+ T Cells

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Human CD4+ T cells and mice CD4+ T cells were purified using negative selection (CD4+ T Cell Isolation Kit, human; CD4+ T cell Isolation kit, mouse; Miltenyi Biotec, Auburn, CA) respectively. Purified human and mice CD4+ T cells were stimulated in the presence of anti-CD3 (5 μg/ml, UCHT1; 1 μg/ml, 145-2C11) and anti-CD28 (1 μg/ml, CD28.2; 1 μg/ml, 37.51) (BD Bio-sciences) for 3d under the following conditions—For human CD4+ T cells: Th1: rhIL-12 (10 ng/ml; Peprotech, Rocky Hill, NJ); Th2: rhIL-4 (25 ng/ml; Peprotech); Th17: TGF-β (5 ng/ml; Peprotech), IL-1β, IL-21 and IL-23 (all 25 ng/ml; Peprotech); Treg: 1 ng/ml rhTGF-β and 2 ng/ml rhIL-2 (Peprotech). Recombinant human IL-2 was bought through ACRO Biosystems and was used at 10 U/ml, except under Treg (2 ng/ml) and Th17 conditions. For mice CD4+ T cells: Th1: 40 ng/ml rIL-12 (Peprotech) and 40 μg/ml anti-IL-4 (R & D); Th2: 40 ng/ml rIL-4 (Peprotech) and 10 μg/ml anti-IFN-γ (R & D); Th17: 1 ng/ml rTGF-β and 40 ng/ml rIL-6 (both Peprotech), 40 μg/ml anti-IL-4 and 10 μg/ml anti-IFN-γ (both R & D); Treg: 1 ng/ml rTGF-β and 2 ng/ml rIL-2 (both Peprotech).
Zhang R., Zeng H., Zhang Y., Chen K., Zhang C., Song C., Fang L., Xu Z., Yang K., Jin B., Wang Q, & Chen L. (2016). CD226 ligation protects against EAE by promoting IL-10 expression via regulation of CD4+ T cell differentiation. Oncotarget, 7(15), 19251-19264.
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7

Cytotoxicity Assay of HIV Drugs

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rhIL-2 and rhIL-15 were provided by the BRB/NCI Preclinical Repository. rhIL-12 was obtained from PeproTech and rhIL-18 from R&D systems. HODHBt was purchased from AK Scientific. K562-green fluorescent protein (GFP) (ATCC CCL-242-GFP) cell culture was maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), glutamine, and penicillin-streptomycin. A2780 cell culture was maintained in RPMI 1640 medium supplemented with 10% FBS, glutamine, and penicillin-streptomycin. U87 cell culture was maintained in DMEM supplemented with 10% heat-inactivated FBS, glutamine, OCILy1 and OCILy10 cell cultures were maintained in Iscove’s modified Dulbecco’s medium (IMDM) medium supplemented with 10% human serum, glutamine, and penicillin-streptomycin. Nelfinavir and raltegravir were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, and HIV-1NL4-3 from Malcolm Martin.
Macedo A.B., Levinger C., Nguyen B.N., Richard J., Gupta M., Cruz C.R., Finzi A., Chiappinelli K.B., Crandall K.A, & Bosque A. (2022). The HIV Latency Reversal Agent HODHBt Enhances NK Cell Effector and Memory-Like Functions by Increasing Interleukin-15-Mediated STAT Activation. Journal of Virology, 96(15), e00372-22.
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8

Characterization of NK Cell Function

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The following anti-human monoclonal antibodies (mAbs) were used: CD56(N901), CD3(UCHT1), CD159a(Z199), CD158a,h(EB6B), and CD45(J.33; all Beckman Coulter); CD16(3G8), CD158b(CH-L), IFNγ(B27), CD107a(H4A3), Syk(4D10), ZAP-70(1E7.2), pZAP70(pY319)/pSYK(pY352), pERK1/2(pT202/pY204), pAkt(pS473; all BD); CD3z(6B10.2; eBioscience); CD158e1(DX9), CD107a(H4A3), goat anti-mouse IgG(Poly4053; all Biolegend); FcεR1γ (Milli-Mark); and rituximab (Genentech). The following endotoxin-free recombinant human (rh) cytokines were used: rhIL-12, rhIL-18 (Peprotech), rhIL-15 (CellGenix and Miltenyi). The following cell lines were used: K562 (ATCC, CCL-243) and Raji (ATCC, CCL-86). Cells were obtained from ATCC in 2008, viably cryopreserved and stored in LN2, thawed for use in these studies, and maintained for <2 months at a time in continuous culture as per ATCC instructions. K562 cells were authenticated in 2015 by confirming cell growth morphology (lymphoblast), short tandem repeat profile, growth characteristics, and functionally as NK cell sensitive targets. Raji cells were authenticated in 2015 by confirming cell growth morphology (lymphoblast), short tandem repeat profile, growth characteristics, phenotype of uniform expression of human CD20, and functionally as anti-CD20 mAb opsonized targets for ADCC.
Wagner J.A., Berrien-Elliott M.M., Rosario M., Leong J.W., Jewell B.A., Schappe T., Abdel-Latif S, & Fehniger T.A. (2016). Cytokine-induced memory-like differentiation enhances unlicensed NK cell anti-leukemia and FcγRIIIa-triggered responses. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation, 23(3), 398-404.
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9

Quantifying IFNγ and CD107a in PBMC

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1 × 106 fresh PBMCs from CHB patients were stimulated with or without recombinant human IL-12 (rhIL-12; 10 ng/mL, Peprotech) for 16 h, and IFNγ production by CD56+ T cells was determined as above. For CD107a detection after stimulation in vitro, 1 × 106 human PBMCs and 1 × 105 cells of the erythroleukemia cell line K562 (American Type Culture Collection) were co-cultured for 4 h. Degranulation was detected with a PE-labelled anti-CD107a.
Guo C., Shen X., Fu B., Liu Y., Chen Y., Ni F., Ye Y., Sun R., Li J., Tian Z, & Wei H. (2016). CD3brightCD56+ T cells associate with pegylated interferon-alpha treatment nonresponse in chronic hepatitis B patients. Scientific Reports, 6, 25567.
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10

Optimized Dendritic Cell Activation Protocol

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As shown in Figure 1B, agents added standardly during Step 1 (d0-2 of culture) included recombinant human (rh) GM-CSF on d0 of culture (sargramostim, Sanofi-Aventis, Bridgewater NJ, optimized final concentration 40 ng/ml); resiquimod on d1 of culture (R848 VacciGrade, Invivogen, San Diego CA, optimized final concentration 3 μg/ml); and LPS on d1 of culture (E. coli 026:B6, Sigma-Aldrich, St Louis MO, #2654, optimized final concentration 5 ng/ml). Other agents tested in Step 1 but absent from the optimized formulation included rhIL-4 (Peprotech, Rocky Hill NJ, #200-04. 20 ng/ml); rhIL-12 (Peprotech #200-12, 10 ng/ml); rhIFNγ (Actimmune, Horizon Pharma, Deerfield IL, 2000 IU/ml); and polyI:C (Sigma-Aldrich #P1530, 50 μg/ml). Ags added on d1 of culture (prior to R848 and LPS) included MUC1-, HER2/neu- and CMVpp65-derived synthetic peptides added at an optimized final concentration of 50 μg/ml when pulsed singly, or 10 μg/ml each when multiple peptides were pulsed as a cocktail (see Peptide synthesis below); clinical grade Candida albicans extract (CAN, Hollister Stier, Spokane WA, #5053, at a 10% v:v optimized final concentration); and recombinant HER2/neu intracellular domain protein (HER2-ICD, SignalChem, Richmond,BC #E27-11G at an optimized final concentration of 50 μg/ml).
Pathangey L.B., McCurry D.B., Gendler S.J., Dominguez A.L., Gorman J.E., Pathangey G., Mihalik L.A., Dang Y., Disis M.L, & Cohen P.A. (2016). Surrogate in vitro activation of innate immunity synergizes with interleukin-7 to unleash rapid antigen-driven outgrowth of CD4+ and CD8+ human peripheral blood T-cells naturally recognizing MUC1, HER2/neu and other tumor-associated antigens. Oncotarget, 8(7), 10785-10808.
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