Mice were infected with a medium dose of FV. Starting from day 0, mice were injected i.p. twice with IL-15 (2.5 μg, recombinant mouse IL-15; BioLegend) and three times with IL-18 (1 μg, recombinant mouse IL-18; R&D Systems).
Recombinant mouse il 18
Manufactured by R&D Systems
Sourced in Switzerland
Recombinant mouse IL-18 is a cytokine produced by R&D Systems. It is a member of the interleukin-1 family and is involved in the regulation of the immune response.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant mouse il 12, by Thermo Fisher Scientific (3 mentions)
Recombinant mouse il 1β, by R&D Systems (2 mentions)
Krn7000 αgc, by Funakoshi (2 mentions)
Tigit blocking mab clone 1b4, by BioXCell (2 mentions)
Recombinant mouse il 15, by BioLegend (1 mentions)
Cd4 l3t4 microbeads, by Miltenyi Biotec (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Recombinant mouse il 17, by R&D Systems (1 mentions)
Golgistop, by BD (1 mentions)
Anti ly49d 4e5, by BD (1 mentions)
Lipopolysaccharide lps, by Enzo Life Sciences (1 mentions)
Hydroxylamine, by Merck Group (1 mentions)
Brefeldin a, by BioLegend (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Halolink resin, by Promega (1 mentions)
Pam3csk4, by InvivoGen (1 mentions)
7 protocols using recombinant mouse il 18
1
Immunotherapy for Retroviral Infection
2
Effector T cell and BMMC co-culture
3
Isolation and Stimulation of Mouse RPE Cells
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All procedures using animals adhered to the Association for Research in Vision and Ophthalmology statement for the use of animals and the NEI's Institutional Animal Care and Use Committee approved protocols. Mouse RPE was isolated from retinas of C57/B6J mice at 6–8 weeks of age as described previously [15 (link)]. Briefly, mice were euthanized, and their eyes were enucleated. The globes were washed with PBS containing 1% penicillin-streptomycin (Sigma) and then were dissected free of periocular connective tissue. Then, the globe was placed on 2% Dispase II (neutral protease, grade II, Roche, Indianapolis, IN, USA) and incubated at 37°C for 40 min. The globe was transferred to DMEM/F12 media, the anterior segment was removed, and the retina containing the RPE layer was dissected free. The loosely adherent RPE cell layer was gently separated from the retina and transferred to a 15 mL tube containing DMEM/F12, 20% FBS, and 1% L-glutamine-penicillin-streptomycin. Cells were then centrifuged at 1000 rpm for 5 min and resuspended. The RPE suspension was added to 6-well cell culture plates. The medium was changed after 5-6 days and every 2-3 days thereafter. The RPE cells between two and three passages were stimulated with 100 ng/mL recombinant mouse IL-1β (R&D Systems), 10 ng/mL recombinant mouse IL-18 (MBL), or 10 ng/mL recombinant mouse IL-17 (R&D Systems) for 24 hours.
4
Cytokine-Activated NK Cell Assay
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3.106 splenic lymphocytes or tumor cell suspensions were prepared in complete medium (RPMI + glutamax, 10% SVF, 1% penicillin/streptomycin, 10 mM HEPES, 1 mM sodium-pyruvate, 50 μM 2-mercaptoethanol) and incubated for 4 h with cytokines [recombinant mouse IL-12 (Peprotech, 200-12) (final concentration: 100 ng/mL) and recombinant mouse IL-18 (R&D, B004-5) (final concentration: 20 ng/mL)] or on antibody-coated plates [anti-NKp46 (29A1; BD Biosciences), anti-Ly49D (4E5; BD Biosciences), anti-NKG2D (CX5; BD Biosciences), and GolgiStop (BD Biosciences) in the presence of anti-CD107a (2B6; BD Biosciences)] or co-cultured with Yac-1 or 4T1 cells (1:1 ratio).
5
Glycolipid Activation of iNKT and MAIT Cells
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Glycolipid stimulation of iNKT cells was achieved via i.p. administration of 100 μg/kg of KRN7000/αGC (Funakoshi, Tokyo, Japan) or 200 μg/kg of αCGC in a vehicle containing 5.6% sucrose, 0.75% L-histidine and 0.5% Tween-20, which was further diluted in PBS. αCGC was supplied by the NIH Tetramer Core Facility (Atlanta, GA). To stimulate iNKT cells in a cytokine-dependent manner, each animal was injected i.v. with 2 ng recombinant mouse IL-12 (Peprotech, Rocky Hill, NJ) plus 200 ng recombinant mouse IL-18 (R&D Systems) in PBS.
To activate MAIT cells, mice were injected i.p. with 200 μL of PBS containing 20 μL of a 5-OP-RU stock solution. The stock solution was prepared by mixing equal volumes of 2 mM 5-amino-6-D-ribitylaminouracil (5-ARU) and 2 mM methylglyoxal in DMSO for 24 h at room temperature. Aliquots were stored at −80°C until use. Control mice received vehicle (2 mM methylglyoxal in DMSO) diluted in PBS.
Where indicated, mice were given 200 μg of a TIGIT-blocking mAb (clone 1B4) or a mouse IgG1κ isotype control (clone MOPC-21 from BioXCell) 1 h prior to αGC administration.
To activate MAIT cells, mice were injected i.p. with 200 μL of PBS containing 20 μL of a 5-OP-RU stock solution. The stock solution was prepared by mixing equal volumes of 2 mM 5-amino-6-D-ribitylaminouracil (5-ARU) and 2 mM methylglyoxal in DMSO for 24 h at room temperature. Aliquots were stored at −80°C until use. Control mice received vehicle (2 mM methylglyoxal in DMSO) diluted in PBS.
Where indicated, mice were given 200 μg of a TIGIT-blocking mAb (clone 1B4) or a mouse IgG1κ isotype control (clone MOPC-21 from BioXCell) 1 h prior to αGC administration.
6
Glycolipid Activation of iNKT and MAIT Cells
7
Preparation and Purification of Protein Phosphatases
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Hydroxylamine (50% wt/vol) was obtained from Sigma-Aldrich (Merck Group, Munich, Germany), the protein phosphatase from bacteriophage k (kPPase) from New England Biolabs (Hertfordshire, UK) and the TLRactivating ligands Pam3CSK4 and R848 (Resiquimod) from InvivoGen. LPS (lipopolysaccharide) was from Enzo Life Sciences (Lausen, Switzerland), recombinant mouse IL-18 from R&D Systems (Minneapolis, MN, USA), Halo-Link Resin from Promega (Fitchburg, WI, USA), Protein G-Sepharose from Expedeon (Cambridge, UK), reagents for cell culture from Gibco Thermo Fischer Scientific (Cambridge, MA, USA), Immobilon-P PVDF membranes from Merck-Millipore (Merck Group) and Brefeldin A (420601) from BioLegend (San Diego, CA, USA). Other solvents and reagents were obtained from Sigma-Aldrich (Merck Group) or VWR International (Radnor, PN, USA). The deubiquitylases USP2 and Otulin were expressed and purified as described [53] .
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