For cytokine production analyses, thawed and rested PBMCs were stimulated with PMA (5 ng/mL; Sigma) plus Ionomycin (500 nmol/L; Sigma), or with human IL12 (10 ng/mL; Pepro-tech) plus human IL18 (50 ng/mL; R&D Systems). Alternatively, PBMCs were exposed to the target cell lines K562 (ATCC CCL-243) or to anti–CD20-coated Raji (ATCC CCL-86) that had been preincubated with 10 mg/mL of the antibody (30 minutes, 4°C) and washed. Stimulation and coincubations were done for 5 hours at 37°C with Brefeldin A present during the last 4 hours. For proliferation assays, sorted cells were carboxyfluorescein succinimidyl ester (CFSE)-labeled and cultured in IL15 at indicated dosages for 7 days.
Human il 12
Manufactured by Thermo Fisher Scientific
Human IL-12 is a recombinant cytokine that plays a central role in the regulation of cell-mediated immunity. It is composed of two subunits, p35 and p40, and is involved in the differentiation of naive T cells into Th1 cells.
Automatically generated - may contain errors
Lab products found in correlation
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Human il 18, by R&D Systems (2 mentions)
Ccl 86, by American Type Culture Collection (1 mentions)
Il 23, by Thermo Fisher Scientific (1 mentions)
Il 35, by Thermo Fisher Scientific (1 mentions)
Human il 2, by Thermo Fisher Scientific (1 mentions)
Human il 15, by Thermo Fisher Scientific (1 mentions)
Easysep human nk cell isolation kit, by STEMCELL (1 mentions)
Nk macs medium, by Miltenyi Biotec (1 mentions)
Human ifn γ, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Anti il 4, by Thermo Fisher Scientific (1 mentions)
Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions)
Na ve cd4 t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
5 protocols using human il 12
1
Cytokine Production and Proliferation Assay
2
Cytokine Production Analyses of PBMCs
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For cytokine production analyses, thawed and rested PBMCs were stimulated with PMA (5 ng/mL, Sigma) plus ionomycin (500 nmol/L, Sigma), or with human IL-12 (10 ng/mL, PeproTech) plus human IL-18 (50 ng/mL, R&D Systems). Alternatively, PBMCs were exposed to the target cell lines K562 (ATCC CCL-243), MHC class I chain-related protein A (C1R-MICA) MHC class I related stress-inducible surface glycoprotein (kindly provided by Dr Veronika Groh-Spies, Fred Hutchinson Cancer Research Center) or to anti-CD20-coated Raji (ATCC CCL-86) cells that had been preincubated with 10 µg/mL of the antibody (30 min at 4°C) and washed. Stimulation and coincubations were done for 5 hours at 37°C with brefeldin A present during the last 4 hours.
3
Measuring IL-12 and IL-35 Interactions
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Human IL-12 (PeproTech, Rocky Hill, NJ; 200-12H), IL-23 (PeproTech, 200-23), and IL-35 (PeproTech, 200-37) were reconstituted in sterile DMEM. To measure reporter cells’ response to IL-12 in the presence or absence of IL-35, ∼50,000 cells were added to each well of a 96-well plate and allowed to settle overnight (37°C, 5% CO2). The next morning, 20 μl IL-12 and/or IL-35 was added at the indicated final concentrations in triplicate (i.e., three biological replicates). IL-23 alone at the same concentrations was included as a control to determine the IL-12 specificity reporter cells. Cells were then incubated for 12 h (37°C, 5% CO2), after which the cell supernatants were used to measure SEAP levels via the QUANTI-Blue solution method (InvivoGen). Absorbance readings were taken (wavelength 630 nm), and dose–response curves were plotted using GraphPad Prism (version 8.0). Each IL-12 ± IL-35 dose–response assay shown was repeated at least three times.
4
Isolation and Cytokine Stimulation of Human NK Cells
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Human PBMCs from anonymous healthy donors were obtained from leukoreduction filters after platelet apheresis from the UCLA Virology Core. NK cells were isolated using the EasySep Human NK Cell Isolation Kit (Stem Cell Technologies) following the manufacturer's instructions. Following isolation, cells were maintained in 24-well G-Rex plates (Wilson Wolf) in NK MACS medium (Miltenyi Biotech) supplemented with human IL-2 (100 IU ml -1 , PeproTech) and human IL-15 (20 ng ml -1 , PeproTech) at a plating density of 5 × 10 6 cells per well. For cytokine stimulation, either freshly isolated human NK cells or cells activated for 14 d with IL-2/IL-15 were plated with K562 leukemia cells at an effector:target ratio of 2.5:1 in addition to human IL-2 (100 IU ml -1 , PeproTech), human IL-15 (20 ng ml -1 , PeproTech), human IL-12 (10 ng ml -1 , PeproTech) and/or human IL-18 (100 ng ml -1 , PeproTech) in CR-10 media. NK cells were stimulated with cytokines for 16 h before analysis by flow cytometry.
5
Naive CD4+ T Cell Polarization
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Untouched naïve human CD4+ T cells were isolated from PMBCs by magnetic-activated cell sorting using the Naïve CD4+ T Cell Isolation Kit II (Miltenyi) and an LS Column (Miltenyi) according to the manufacturer's protocol. TH1 T cell polarization was induced by using 5 µl Dynabeads Human T-Activator CD3/CD28 (Gibco) with 1×106 cells, 10 ng/ml human IFN-γ (Peprotech), 10 ng/ml human IL-12 (Peprotech) and 1 µg/ml anti-IL-4 (Peprotech). T cells were cultured for 14 days in RPMI medium containing L-glutamine and 25 mM HEPES (Gibco) supplemented with 10% FCS, 1% penicillin-streptomycin (Thermo Fisher Scientific) and 0.02 mM sodium pyruvate (Thermo Fisher Scientific) at 37°C and 5% CO2.
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