The expansion of isolated pNK cell was performed with a GMP-compliant method at CHA Biotech (Seongnam, Korea). The cells were seeded on a γ-globulin (Greencross, Yongin, Korea) and anti-NKp46 (R&D Systems, Minneapolis, MN, USA)-coated flask and cultured in Alys505NK serum-free medium (CSTI, Sendai, Japan) supplemented with 1000 IU/mL recombinant human IL-2 (Novartis, Basel, Switzerland), 50 ng/mL recombinant human IL-18 (R&D system, Minneapolis, MN, USA), and 5% heat-inactivated autologous plasma. Fresh culture medium was added every 1 to 3 days depending on the cell density (2 × 106 cells/mL). On Day 6, the cells were transferred to a culture bag (NIPRO, Osaka, Japan), and cultured for 14 days. The pNK cells were cryopreserved with Cryostor CS5 (BioLife Solutions, Bothell, WA, USA).
Recombinant human il 18
Manufactured by R&D Systems
Sourced in Switzerland, United States
Recombinant human IL-18 is a cytokine that belongs to the IL-1 family. It is produced in E. coli and purified. The recombinant protein has a molecular weight of approximately 18 kDa.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant human il 2, by Novartis (1 mentions)
Anti nkp46, by R&D Systems (1 mentions)
Cryostor cs5, by BioLife Solutions (1 mentions)
Cd56 pe, by Beckman Coulter (1 mentions)
Recombinant human il 12, by R&D Systems (1 mentions)
Fixable viability dye ef780, by Thermo Fisher Scientific (1 mentions)
Ifn γ fitc clone 4 s b3, by BioLegend (1 mentions)
Rhtnf α, by Thermo Fisher Scientific (1 mentions)
Rhil 15, by Thermo Fisher Scientific (1 mentions)
Rhil 18, by R&D Systems (1 mentions)
Rhil 6, by Miltenyi Biotec (1 mentions)
Round bottom 96 wells plates, by Greiner (1 mentions)
Recombinant human il 2, by BD (1 mentions)
Leaf anti human mouse rat mr1, by BioLegend (1 mentions)
Rat igg2a, by R&D Systems (1 mentions)
Vaccinia virus b18r carrier free recombinant protein, by Thermo Fisher Scientific (1 mentions)
Mouse igg1, by R&D Systems (1 mentions)
5 protocols using recombinant human il 18
1
Expansion of Purified Natural Killer Cells
2
PBMC Cytotoxicity Assay with IL-12/IL-18 Stimulation
3
Stimulation of NK Cells with B. pertussis
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NK cells were seeded into round-bottom 96-wells plates (Greiner) at 150,000 cells/well in IMDM culture medium supplemented with 5 ng/ml recombinant human IL-15 (rhIL-15; PeproTech) (29 (link)). Next, NK cells were immediately stimulated with B. pertussis B4393 at a MOI of 10 in the presence or absence of 5 ng/ml recombinant human IL-18 (rhIL-18; R&D systems), 10 ng/ml rhIL-6 (Miltenyi Biotec), 10 ng/ml rhTNFα (PeproTech), or 10 ng/ml rhIL-1β (InvivoGen). Stimulations were performed at 37°C and 5% CO2 for 18 h after which BD GolgiPlugTM containing Brefeldin A (BD Biosciences) was added to the culture for 4 h, to inhibit cytokine secretion, before collecting the NK cells for flow cytometry analysis.
4
Cytokine-Induced IFN-γ Release Assay
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Human whole blood was incubated with deucravacitinib, tofacitinib, upadacitinib, or baricitinib for 1 h at 37 °C prior to being stimulated with 2 ng/mL of recombinant human IL-12 (catalog no. 200-12; PeproTech US) plus 10 ng/mL of recombinant human IL-18 (catalog no. 9124-IL-050; R&D Systems, Inc, Minneapolis, MN) overnight at 37 °C in a carbon dioxide incubator. Blood was centrifuged at 1200 rpm for 10 min, and plasma was removed for analysis of IFN-γ levels using a BD OptEIA human IFN-γ enzyme-linked immunosorbent assay kit (catalog no. 555142; BD Biosciences).
The IC50 values, as well as Hill coefficients for inhibition, were determined for deucravacitinib, tofacitinib, upadacitinib, and baricitinib using these assays.
The IC50 values, as well as Hill coefficients for inhibition, were determined for deucravacitinib, tofacitinib, upadacitinib, and baricitinib using these assays.
5
Optimizing Cytokine-Mediated Whole Blood Stimulations
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Cytokines used for whole blood stimulations included the following: recombinant human IL-2 (BD Biosciences), recombinant human IL-12p70 (eBioscience), recombinant human IL-18 (R&D Systems), and rIFN (eBioScience), each at a final concentration 0.1 μg/ml or lower, as determined by cytokine titration experiments. Neutralizing Abs included rat anti-human IL-2 (clone MQ1-17H12; BD Biosciences), mouse anti-human IL-12 (clone 24910; R&D Systems), mouse anti-human IL-18 (clone 125-2H; R&D Systems), LEAF anti-human/mouse/rat MR1 (catalog no. 361103; BioLegend, San Diego, CA), and mouse anti-human TCRα (clone T10B9.1A-31; BD Biosciences); the isotype controls included mouse IgG1 (clone 11711; R&D Systems) and rat IgG2a (clone 54447; R&D Systems). For type I IFN neutralization, we used vaccinia virus B18R Carrier-Free Recombinant Protein (eBioScience). All neutralizing Abs were used at a final concentration 10 μg/ml.
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