Special agents used in this study were as follows: recombinant human AR (Wako Chemicals USA, Inc., Richmond, VA), rabbit anti-human AR Ab (Santa Cruz Biotechnology Inc., Santa Cruz, CA), horseradish peroxidase-conjugated anti-phosphotyrosine mAb (clone RC20) (BD Transduction Laboratories, Fraklin Lakes, NJ), anti-phosphoserine mAb (Calbiochem-Novabiochem Corporation, San Diego, CA), rabbit anti-p44/42 MAP kinase Ab (Cell Signaling Technology, Inc., Danvars, MA), rabbit anti-phospho-p44/42 MAP kinase Ab (Thr202/Thr204) (Cell Signaling Technology, Inc.), rat anti-mouse CD3 mAb (Serotec Ltd., Oxford, UK), hamster anti-mouse CD28 mAb (Pharmingen Co., San Diego, CA), DynabeadsR mouse CD3/CD28 T cell expander (Invitrogen Dynal AS, Oslo, Norway), rat anti-mouse IL-2 mAb (R & D systems, Inc., minneapolis, MN), rat anti-mouse IFN-γ mAb (R & D systems), mouse IL-2 (R & D systems), mouse IFN-γ (R & D systems), D10.G4.1 T cell line (TIB 224; American Type Culture Collection (ATCC), Rockville, MD), epalrestat (Wako Pure Chemicals, Osaka, Japan), DL-glyceraldehyde (Wako), β-NADPH (Wako), imidazole (Wako), [3H]-thymidine (3H-TdR) (PerkinElmer Life Science Products Inc., Boston, MA).
Mouse il 2
Manufactured by R&D Systems
Sourced in United States
Mouse IL-2 is a recombinant protein that represents the interleukin-2 cytokine from the mouse. Interleukin-2 is a signaling molecule that plays a key role in the activation and proliferation of T cells, which are important for immune function.
Automatically generated - may contain errors
Lab products found in correlation
Human tgf β1, by R&D Systems (4 mentions)
Anti il 4 neutralizing antibody, by Thermo Fisher Scientific (3 mentions)
Anti cd3, by Thermo Fisher Scientific (3 mentions)
Soluble anti cd28, by Thermo Fisher Scientific (3 mentions)
Anti ifnγ neutralizing antibody, by Thermo Fisher Scientific (3 mentions)
Anti il 12 23 p40 neutralizing antibody, by Thermo Fisher Scientific (3 mentions)
Mouse il 6, by R&D Systems (3 mentions)
Human il 2, by R&D Systems (2 mentions)
Anti il 12 23 p40 neutralizing antibody, by R&D Systems (2 mentions)
Naive cd4 t cells isolation kit, by Miltenyi Biotec (2 mentions)
Mouse il 27, by R&D Systems (2 mentions)
Recombinant dll4, by R&D Systems (2 mentions)
Rat anti mouse ifn γ mab, by R&D Systems (1 mentions)
Dl glyceraldehyde, by Fujifilm (1 mentions)
β nadph, by Fujifilm (1 mentions)
Imidazole, by Fujifilm (1 mentions)
Mouse ifn γ, by R&D Systems (1 mentions)
Mouse pan t cell isolation kit, by Miltenyi Biotec (1 mentions)
Human pan t cell isolation kit, by Miltenyi Biotec (1 mentions)
Anti cd3 anti cd28 dynabeads, by Thermo Fisher Scientific (1 mentions)
13 protocols using mouse il 2
1
Antibody-mediated Immune Cell Activation
2
Activated Primary T Cell Proliferation Assay
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Assays with human primary T cells were conducted by Pharmaron. Human peripheral blood mononuclear cells for these assays were sourced commercially from Sailybio (Cat. No. SLB-HP200A), with ethical approvals and informed consent for the collection. Human primary T cells were isolated from fresh peripheral blood mononuclear cells (Sailybio) using the human Pan T Cell Isolation Kit (Miltenyi Biotec) and resuspended in RPMI medium 1640 containing 10% FBS, 1% penicillin/streptomycin, and 10 ng/mL human IL-2 (R&D Systems). Mouse primary T cells were isolated from fresh spleen cells using the mouse Pan T Cell Isolation Kit (Miltenyi Biotec) and resuspended in RPMI medium 1640 containing 10% FBS, 1% penicillin/streptomycin, and 10 ng/mL mouse IL-2 (R&D Systems). Compounds R80 and T35 were added as appropriate to various concentrations. Cells were seeded into 96-well plates and incubated at 37 °C and 5% CO2 for 1 h. Human or mouse anti-CD3/anti-CD28 Dynabeads (Thermo Fisher Scientific) (78 (link)) and 100 μM cytidine (where appropriate) were then added, and cells were incubated at 37 °C and 5% CO2 for 5 d. CTG reagent (Promega) was added to cells and incubated at room temperature for 30 min, after which luminescence was recorded using a Perkin-Elmer Envision microplate reader.
3
Differentiation of Naive CD4+ T Cells
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Naïve CD4+ T cells were isolated from the spleens of WT mice or AhR-/- mice using the Naive CD4+ T-cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany). For Treg cell polarization, naive CD4+ T cells were stimulated with immobilized anti-CD3 mAb (2 μg/mL, clone145-2G1, eBioscience) and soluble anti-CD28 mAb (1 μg/mL, clone37.51, eBioscience) supplemented with human TGF-β1 (2.5 ng/mL, R&D Systems) and mouse IL2 (10 ng/mL, R&D Systems) with the indicated concentration of mesalamine for 3 days. For Th1 or Th17 cell polarization, naive CD4+ T cells were stimulated with immobilized anti-CD3 mAb and soluble anti-CD28 mAb for Th0; with recombinant mouse IL12 (10 ng/mL, R&D Systems) plus anti-IL4 mAb (10 μg/mL, clone11B11, eBioscience) for Th1; and with human TGF-β1, mouse IL6 (30 ng/mL, R&D Systems), anti-IL4 mAb, and anti-IFN-γ mAb (10 μg/mL, cloneXMG1.2, eBioscience) for Th17, with the indicated concentration of mesalamine, for 3 days.
4
Differentiation of Naive T Cells into Th Subsets
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CD4+ CD25−CD62LhiCD44lo naive T cells were enriched from spleen using the naive CD4 T cells isolation kit (Miltenyi Biotec) with more than 92% purity. Naive T cells were then plated and cultured in 24-well plates. Naive T cells (106/0.5 mL) were stimulated with plate-bound anti-CD3 (2.5 μg/mL; eBioscience), soluble anti-CD28 (3 μg/mL; eBioscience), and plate-bound recombinant DLL4 (1.65 μg/mL, R&D). In addition, recombinant cytokines and neutralizing antibodies were added to skew toward different Th cells in vitro. For Th1: mouse IL-12 (10 ng/mL), anti-IL-4 neutralizing antibody (10 μg/mL; eBioscience); for Th2: mouse IL-4 (10 ng/mL; R&D System), anti-IFNγ neutralizing antibody (10 μg/mL; eBioscience), anti-IL-12/23 p40 neutralizing antibody (10 μg/mL); for Th17 cells: mouse IL-6 (10 ng/mL; R&D System), human TGF-β1 (2 ng/mL; R&D System), anti-IFNγ neutralizing antibody (10 μg/mL; eBioscience), anti-IL-4 neutralizing antibody (10 μg/mL; eBioscience), and anti-IL-12/23 p40 neutralizing antibody (10 μg/mL; eBioscience) were added; for IL-27-inducing TR1, mouse IL-27 (20 ng/mL, R&D) were added; to skew toward in vitro-iTreg cells (iTreg), human TGFβ1 (2 ng/mL; R&D System) and mouse IL-2 (10 ng/mL; R&D System) were added at the same time.
5
Cytokine Secretion Profiling
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At indicated time points of cell culture, supernatants were collected and preserved at −20 °C. Concentrations of cytokines in culture supernatants were determined by using mouse IL-2, IL-10, IL-17A and IFN-γ ELISA kits according to the manufacturer’s instructions (all from R&D Systems, Minneapolis, MN, USA).
6
Differentiation of Naive T Cells into Th Subsets
7
In Vitro Cytokine Profiling Assay
8
Splenocyte Activation Cytokine Profiling
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Splenocytes were cultured for 72 h at 37°C in fresh media at a density of 1×106 cells/well in 12-well anti–CD3-coated tissue culture plates with 30 IU mouse IL-2 (R&D Systems, Minneapolis, MN). During the last 6 h of culture, the cells were stimulated with 1X cell stimulation cocktail (Thermo Fisher Scientific, Waltham, MA, USA). After stimulation, supernatants were collected for quantitation of inflammatory cytokines using the Mouse Cytokine Array/Chemokine Array 31 Plex Assay (MD31, Eve Technologies, Calgary, Alberta, Canada).
9
Generation and Expansion of CD8+ T Cells
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Con-CD8 and Cap-CD8 T cells were generated as previously published27 (link) with the following modifications. Male BALB/c mice (6–8 weeks old; Jackson Laboratory) received three subcutaneous injections every 4 days consisting of 1,000 IU mouse IL-2 (R&D Systems), 25 μg of TLR9 activator ODN 1826 (InvivoGen), and 10 μg of the control AH1 gp70 epitope SPSYVYHQF or dominant capsid CD8+ T cell epitope VPQYGYLTL emulsified in Sigma Adjuvant System (Sigma). Splenocytes were isolated 12 days following the first immunization, and Cap-CD8 were expanded in vitro. A total of 2 × 107 splenocytes was cultured in 6 mL of stimulation media over a 6-day period at 37°C in 15-mL Falcon tubes (BD Biosciences) with media exchange on day 3. Stimulation medium was RPMI-1640 (Invitrogen) supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin, 10 μg/mL SPSYVYHQF or VPQYGYLTL, 25 ng/mL IL-15, and 10 ng/mL IL-21. Following in vitro expansion, CD8+ T cells were magnetically purified using a mouse CD8− selection kit (Miltenyi) and used for in vivo studies.
10
Ex Vivo Enrichment and Activation of NK Cells
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NK cells were enriched ex vivo using a no-touch bead enrichment protocol as previously described (21) , and plated at 10 5 per well with mouse IL-2, IL-12, or IL-15 (R&D Systems) at 20 ng/ml, IL-18 (MBL) at 10 ng/ml, or IFN-α (R&D Systems) at 100 IU. Intracellular staining for IFN-γ was performed as previously described (21) .
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