Naïve human cells were isolated by using Human Naïve CD4+ T Cell isolation kit (Stemcell Technologies, 17555) from peripheral blood mononuclear cells (PBMCs) after Ficoll-Paque gradient (GE Healthcare Systems). Naïve human CD4+ T cells were incubated with Dynabeads™ Human T-Activator CD3/CD28 (ThermoFisher, 11161D) for 3 days in R10 under the following skewing conditions: a) rhIL-23 (25 ng/ml, PeproTech); b) rhIL-23 (25 ng/ml, PeproTech) plus rhIL-10 (10 ng/ml PeproTech); c) rhIL-12 p70 (1 ng/ml, PeproTech) and d) rhIL-12 p70 (1 ng/ml, PeproTech) plus rhIL-10 (10 ng/ml PeproTech). The former conditions were cultured in the presence/absence of hTGF-β (5 ng/ml) (R&D Systems).
Rhil 23
Manufactured by Thermo Fisher Scientific
Sourced in France, United States
RhIL-23 is a recombinant human interleukin-23 protein. Interleukin-23 is a cytokine that plays a role in immune system regulation.
Automatically generated - may contain errors
Lab products found in correlation
Rhil 1β, by Thermo Fisher Scientific (3 mentions)
Rhil 7, by Thermo Fisher Scientific (2 mentions)
Rhil 15, by Thermo Fisher Scientific (2 mentions)
Rhil 6, by Thermo Fisher Scientific (2 mentions)
Complete rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Rhtgf β, by Thermo Fisher Scientific (1 mentions)
Rhil 21, by Thermo Fisher Scientific (1 mentions)
Human na ve cd4 t cell isolation kit, by STEMCELL (1 mentions)
Htgf β, by R&D Systems (1 mentions)
Rhil 12 p70, by Thermo Fisher Scientific (1 mentions)
Rhil 10, by Thermo Fisher Scientific (1 mentions)
Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions)
Rhil 1β, by R&D Systems (1 mentions)
Anti cd28 mab, by BioLegend (1 mentions)
Rhtgf β1, by Thermo Fisher Scientific (1 mentions)
Anti cd3 mab, by BioLegend (1 mentions)
5 protocols using rhil 23
1
Naïve Human T Cell Differentiation under Cytokine Skewing
2
Cytokine-Mediated PBMC Activation
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Human donor PBMCs (2×106/ml) were cultured in complete RPMI 1640 medium (GibcoBRL) and 10% FCS for 10 days in the presence of α-GalCer along with rhIL-2 (10 ng/ml) in addition to the following cytokines added individually or in combination: rhIL-7 (10 ng/ml), rhIL-15 (10 ng/ml), rhIL-1β (5 ng/ml), rhIL-23 (10 ng/ml) (all from PeproTech).
3
Antigen-Specific T Cell Generation
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Antigen specific T cells were generated by stimulation of the lymphocyte population with peptide pulsed moDCs at a T cell to antigen presenting cell (APC) ratio of 5:1. Where indicated, starting lymphocyte populations were enriched for CD4+ T cells by immunomagnetic negative selection (EasySep Stem Cell Technologies). Cells were co-cultured in cell growth medium supplemented with either standard T cell growth cytokines of 10 ng/ml rhIL-7 and rhIL-15 (Peprotech) or enhanced cytokine conditions of 20 ng/ml rhIL-1β, 20 ng/ml rhIL-6, 10 ng/ml rhIL-7, 10 ng/ml rhIL-15, 50 ng/ml rhIL-21, 25 ng/ml rhIL-23, and 5 ng/ml rhTGFβ (Peprotech). After 72 h of culture, 30 IU/ml rhIL-2 was added to the culture. Cultures were fed or split every 2–3 days as needed. Cultures were re-stimulated up to 2 times with peptide pulsed moDCs every 10–14 days.
4
Dendritic Cells Modulate Th17 Responses
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Dendritic cells (10,000 cells/200 μl/well) were cocultured with autologous CD4+ T cells at a ratio of 1:10 in U-bottom 96-well plates and stimulated with (20 μg/ml) γ-irradiated M. tuberculosis or M. tuberculosis-derived cytoplasmic fractions (10 μg/ml) either alone or in the presence of (10 ng/ml) rhIL-1β (R&D systems, Lille, France) or rhIL-23 (PeproTech, Neuilly-Sur-Seine, France) for 5 days. After 5 days, cell-free supernatants were collected, and T cells were analyzed for Th17 responses by intracellular staining as described earlier.
5
Th17 Cell Differentiation in PBMCs
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To generate Th17 cells in PBMCs, anti-CD3 mAb (0.5 ng/L; BioLegend, USA), anti-CD28 mAb (0.5 ng/L; BioLegend, USA), recombinant human IL-1β (rhIL-1β; 100 ng/mL), rhIL-23 (100 ng/mL), rhIL-6 (100 ng/mL), and rhTGF-β1 (25 ng/mL) (all cytokines from PeproTech, USA) were added to the medium on day 0. When 1,25(OH)2D3 (Selleck Chemicals, USA) was used to treat the cells, a final concentration of 100 nM was employed according to the results of our pre-experiments (not shown). After 72 hours of culture, supernatants were collected, and IL-17A production in culture supernatants was analyzed by enzyme-linked immunosorbent assay (ELISA) (Dakewe Biotech Company, China) according to the manufacturer’s instructions.
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