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2 protocols using mouse il 2

1

Immunoregulatory Mechanisms in Allergic Responses

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ImjectTM Alum Adjuvant was obtained from Pierce Biotechnology (Rockford, IL, USA). MitoSOXTM Red was purchased from Thermo Fisher Scientific (Rockford, IL, USA). Mouse IL-2, IL-4, IL-5, IL-12, interferon (IFN)-γ, and IgE ELISA kits were purchased from BD Biosciences (San Diego, CA, USA). IL-13 ELISA kit was obtained from R&D Systems (Minneapolis, MN, USA). Specific primary antibodies for signal transducer and activator of transcription 6 (STAT6), p21, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibody for phospho-STAT6 was obtained from Abcam (Cambridge, UK). Antibodies for phospho-STAT1, STAT1, phospho-STAT6, STAT6, phospho-ERK1/2, ERK1/2, phospho-p38, p38, phospho-JNK, JNK, NRF2, and HO-1 were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse anti-CD3 and anti-CD28 antibodies were purchased from Biolegend (San Diego, CA, USA). SP600125 was purchased from Calbiochem (San Diego, CA, USA). U0126 was obtained from Promega (Madison, WI, USA) and 2′,7′-Dichlorofluorescein-diacetate (DCFH-DA) was purchased from Invitrogen (Carlsbad, CA, USA). Propidium iodide (PI), SB203580, TMA, ovalbumin (OVA) (Grade VI), dexamethasone (Dex), concanavalin A (Con A), N-acetylcysteine (NAC), and MQL were purchased from Sigma-Aldrich (St. Louis, MO, USA).
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2

Antigen-Specific T Cell Activation by PS-Loaded PLGA Nanoparticles

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Mouse T cells were purified from spleens of 2D2 mice, a genetically engineered mouse model with only T cells expressing T cell receptors to MOG, using anti-CD4+ beads (MACS) [45 (link)]. Bone-marrow derived DCs were incubated with PS-loaded PLGA nano-particles (12, 25, or 50 μM PS), PS alone (12, 25, or 50 μM), or blank PLGA nanoparticles at 37 °C for 4 h, and the cells were subsequently washed. For myelin-specific T cell activation, 5 × 104 DCs treated with PS-loaded PLGA nanoparticles, PS alone, or blank PLGA nanoparticles at 37 °C for 4 h were pulsed with MOG35–55 peptide (50 μg/ml) and then incubated with 2 × 105 T cells for 48 h. The cells and media were separated, and the supernatant was probed for mouse IFNγ (Biolegend, 430806), mouse IL-2 (BD, 555148), mouse IL-6 (BD, 555240) mouse TNFα (BD, 555268). T-cells were stained with IL-10 (eBioscience, 12-7101) and FOXP3 (eBioscience, 17-5773-82) to identify the Treg population using flow cytometry.
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