BMDMs were prepared as described previously (63 (link)). Briefly, bone marrow cells were isolated and grown in IMDM media supplemented with 10% FBS in the presence of 20 ng/ml M-CSF (PeproTech) or 5 ng/ml GM-CSF (PeproTech) for 7 days. BMDMs were pretreated with cp-TLR-i for 1 h and activated with 200 ng/ml LPS (Sigma) for the indicated times. For cocultivation with T cells, CD4 T cells were purified from the spleen using anti-CD4 microbeads and MACS column (Miltenyi Biotec). For Th1 polarization, M-CSF-derived BMDMs were treated with 200 ng/ml LPS for 6 h, washed twice with fresh IMDM containing 10% FBS, and cocultured with CD4 T cells in the presence of 2.5 μg/ml anti-CD3ε (BD Biosciences). GM-CSF-driven BMDMs were activated with LPS and cocultured with CD4 T cells in the presence of anti-CD3ε and neutralizing antibodies against IL-12 and IFN-γ (BD Biosciences) to induce Th17 polarization. Four days after the cocultivation, CD4 T cells were restimulated with 50 ng/ml PMA and 1 μg/ml ionomycin for 6 h or with 0.5 μg/ml anti-CD3e and 0.5 μg/ml CD28 for 1 day for intracellular cytokine staining and cytokine beads array, respectively.
Anti cd3ε
Manufactured by BD
Sourced in United States
Anti-CD3ε is a monoclonal antibody that binds to the CD3 epsilon chain of the T cell receptor complex. It is used in various biomedical research applications to activate and stimulate T cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd28, by BD (15 mentions)
Soluble anti cd28, by BD (8 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (4 mentions)
Golgistop, by BD (3 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Anti cd28 antibody, by Thermo Fisher Scientific (2 mentions)
Mouse cd4 t cell isolation kit, by Miltenyi Biotec (2 mentions)
Ovalbumin 323 339 ova, by Merck Group (2 mentions)
Anti ifn γ, by BD (2 mentions)
Anti cd28 antibody, by BD (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
V450 proliferation dye, by Thermo Fisher Scientific (2 mentions)
Anti cd4, by BD (2 mentions)
Facsaria 3 cell sorter, by BD (2 mentions)
Rhtgf β1, by Thermo Fisher Scientific (2 mentions)
Rmil 12, by R&D Systems (2 mentions)
Rmil 6, by R&D Systems (2 mentions)
Rmil 2, by R&D Systems (2 mentions)
Rmil 1β, by R&D Systems (2 mentions)
C di amp, by InvivoGen (2 mentions)
41 protocols using anti cd3ε
1
Differentiation and Polarization of Bone Marrow-Derived Macrophages
2
Isolation and Stimulation of Anergic T Cells
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For anergic cell induction, spleens were harvested from NOD.Ocabfl/flCD4-cre or control NOD.Ocabfl/fl animals. Single-cell suspensions were generated by grinding and passage through 70-µm strainers. Cells were isolated using a mouse CD4+ T cell isolation kit (Miltenyi Biotec) and stimulated with 5 µg/ml plate-bound anti-CD3ε (BD Bioscience) ± 2 µg/ml anti-CD28 antibody (eBioscience) as described previously (Kim et al., 2019 (link)). For OCA-B inhibitor peptide treatments, cells were isolated from WT C57BL/6 spleens using a mouse naive CD4+ T cell isolation kit (Miltenyi Biotec) and cultured as described previously (Shakya et al., 2011 (link)). Indicated concentrations of peptides were incubated with cells, with media changes every 2 d after primary stimulation. Activation of C57BL/6 CD4+ cells and subsequent profiling of anergic responses were performed identically to the procedure by (Kim et al.,2019 (link)) For peptide restimulation, total WBCs from PLNs were incubated with 10 µg/ml specific peptide for 4 h in the presence of brefeldin A. The peptides used were as follows: GAD65206-214, TYEIAPVFV; GAD65546-554, SYQPLGDKV; IGRP21-29, TYYGFLNFM; IGRP206-214, VYLKTNVFL; IGRP225-233, LRLFGIDLL; IGRP241-249, KWCANPDWI; and IGRP324-332, SFCKSASIP. Cells were then fixed and stained for flow cytometry.
3
Activation of CD4 T Cells with Anti-CD3/CD28 in the Presence of Salp15
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CD4 T cells were purified from the spleens of C57BL/6 mice by negative selection using a CD4 T cell isolation Kit (Miltenyi Biotec, Bergisch Gladbach, GE) according to the manufacturer’s instructions. Purified CD4 T cells at the indicated concentrations were activated with 5 μg/ml of plate-bound anti-CD3ε and 1 μg/ml of soluble anti-CD28 (BD Biosciences, San Diego, CA) in the presence of the indicated concentration of Salp15 or Salp15ΔP11. Cells were incubated at 37 °C in TexMACS Medium (Miltenyi Biotec).
4
T Helper Cell Differentiation Protocol
5
T Helper Cell Differentiation Protocol
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FACS-sorted CD4+CD25− T cells purified from spleen were activated with plate-bound anti-CD3ε (1μg/ml) and anti-CD28 (10μg/ml; 37.51; BD) and cultured for 3 days under TH9 conditions (murine IL-4: 100 ng/ml; human TGFβ: 5ng/ml; and anti–IFNγ: 10 μg/ml), TH2 conditions (murine IL-4; and anti–IFNγ), TH1 conditions (murine IL-12: 1 ng/ml), TH17 conditions (human TGFβ: 1 ng/ml; murine IL-6: 50 ng/ml; anti–IFNγ; and anti–IL-4: 10 μg/ml), and iTreg conditions (human TGFβ: 5ng/ml). Recombinant proteins were purchased from Peprotech and R&D. In some experiments Ovalbumin 323–339 (OVA; Sigma) was used.
6
Wnt Signaling Activation in Hematopoietic Stem Cells
7
Isolation and Activation of Murine T Cells
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Spleens were isolated from control BALB/c mice under aseptic conditions and prepared for cellular suspensions through a 70 μm cell strainer (BD Biosciences, San Jose, CA, USA). Single-cell suspensions were resuspended in an erythrocyte-lysing buffer (BD Biosciences) and washed. T cells were enriched by a Pan T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s specifications. The purity (CD3ε+) was confirmed at > 90% by flow cytometry. Normal T cells were stimulated with 3 μg/ml anti-CD3ε plus 500 ng/ml anti-CD28 antibodies (BD Biosciences) and cultured in RPMI 1640 (Life Technologies) without l -arginine for 24 h. Media was supplemented with 4% fetal bovine serum (Gibco, Carlsbad, CA, USA), 100 U/ml penicillin and 100 μg/ml streptomycin.
8
Th0, Th1, and Th17 Cell Generation
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For Th0 conditions, sorted naïve CD4+ T cells were stimulated (day 0) with plate-bound anti-CD3ε (1 μg/ml; BD Biosciences) and anti-CD28 (3 μg/ml; BD Biosciences) on 48-well-plates (0.3 × 106 cells/well) in 1 ml T cell medium/well (RPMI1640 supplemented with 10% FBS [Sigma/biowest], antibiotics, 50 mM β-mercaptoethanol) supplemented with 20 U/ml recombinant human IL-2 (rhIL-2) (Peprotech) for 3 days, unless otherwise stated. For the assessment of cell proliferation, 1–10 × 106 naïve CD4+ T cells were labeled using Cell Proliferation Dye eFluor™ 450 (Thermo Scientific) according to the manufacturer's protocol prior to activation. Th1 and Th17 cells were generated from sorted naïve CD4+ T cells activated with anti-CD3ε/anti-CD28 for 3 days in the presence of 20 U/ml rhIL-2 (Peprotech), 5 ng/ml IL-12 (R&D Systems) and 3 μg/ml anti-IL-4 (BioXcell) for Th1 cells, and in the presence of 2 ng/ml TGFβ, 10 μg/ml IL-6, 10 μg/ml IL-1α, and 10 μg/ml IL-1β for Th17 cells. For cytokine detection, activated cells were restimulated for 4 h with phorbol 12-myristate 13-acetate (PMA, 25 ng/ml) and ionomycin (Iono, 750 ng/ml) (both from Sigma-Aldrich) in the presence of GolgiStop (BD Biosciences).
9
Immunohistochemical Staining of Mouse Brain
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Mice were anesthetized with a lethal dose of sodium pentobarbital and trans-cardially perfused with PBS. For frozen sections, mice were subsequently perfused and brains were isolated in 4% paraformaldehyde (PFA) followed by 20% sucrose and then flash frozen in liquid nitrogen. 10 μm horizontal or sagittal sections were cut and mounted on Superfrost Plus (Fisher Scientific) glass slides. For paraffin sections, fresh brains were transferred into 10% phosphate-buffered formalin followed by 70% EtOH and paraffin embedding. 5 μm sections were mounted onto glass slides. Sections were de-paraffinized and antigen retrieval was performed in sodium citrate buffer. Sections were incubated with 3% hydrogen peroxide and blocked in 5% serum for 1 hr at room temperature (RT). Slides were incubated with primary antibody (anti-CD3ε [BD Biosciences], anti-CD45R/B220 [BD Biosciences], or anti-VSV(G) [a gift from Dr. E. Brown, University of Ottawa]) for 2 hr, followed by a biotinylated secondary antibody (BD Biosciences) for 30 min at RT. Avidin-Biotin-Peroxidase (ABC Kit, Vector Laboratories) was applied as per the manufacturer’s protocol. 3,3'-Diaminobenzidine (DAB; Sigma) was added for 2 min before immersing in ddH2O and counterstaining with hematoxylin, followed by slide mounting in xylene medium. Sections were scanned using a UMAX Astra 1220S scanner.
10
Quantifying Cytokine Production in LPL
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To measure spontaneous cytokine production by LPL, LPL were cultured in 96-well flat-bottom plate (Falcon; BD Biosciences) without any stimulation for 24 h at 37 °C under 5% CO2. To measure cytokine production by LPL-T cell, isolated LPL was incubated for 48 h in vitro in 96-well flat-bottom plate coating with anti-CD3ε (10 μg/ml) and soluble anti-CD28 (1 μg/ml) antibodies (BD Biosciences, San Diego, CA). The culture supernatants were then harvested and assayed for cytokine concentration with ELISA kits (R&D Systems) according to the manufacturer’s instruction.
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