Retrovirus-mediated gene overexpression of Sox5 or Stat3 in Th17-polarized cells was performed by a RetroNectin-bound virus infection method30 (link),54 (link). In brief, we constructed the retrovirus vectors which expressed Sox5 and Stat3 genes. Then 48-well plates were coated with RetroNectin (25 µg/ml) and anti-CD3 mAb (BioXCell) overnight at 4 °C. Medium containing retrovirus was added to the RetroNectin-coated plate and the plate was centrifuged for 2 h at 2000×g at 32 °C. After washing with PBS, Th17-polarized cells were added to the retrovirus-bound RetroNectin/anti-CD3 mAb-coated plates in the presence of anti-CD28 mAb (2 µg/ml, BioXCell, D665) and were cultured for 48 h at 37 °C. The cells were then harvested for the analysis of target gene expression.
Anti cd3 mab
Manufactured by BioXCell
Sourced in United States
Anti-CD3 mAb is a monoclonal antibody that binds to the CD3 protein complex on the surface of T cells. It is a widely used tool in immunological research and applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd28 mab, by BioXCell (3 mentions)
Retronectin, by BioXCell (1 mentions)
Propionate, by Merck Group (1 mentions)
Butyrate, by Merck Group (1 mentions)
Anti mouse cd4 magnetic particles, by BD (1 mentions)
Soluble anti cd28 mab, by BioLegend (1 mentions)
Celltrace violet proliferation kit, by Thermo Fisher Scientific (1 mentions)
Il 17a, by Thermo Fisher Scientific (1 mentions)
Tgf β, by R&D Systems (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
5 protocols using anti cd3 mab
1
Retroviral Overexpression of Th17 Regulators
2
Activation of Mouse CD4+ T Cells
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Mouse CD4+ T cells were isolated from spleen or mesenteric lymph node (MLN) using anti-mouse CD4 magnetic particles (Cat#551539, BD Biosciences). CD4+ T cells were seeded in the 24-well plates, and activated with 5 µg/ml anti-CD3 mAb (Clone#145-2C11, Cat#BE0001-1, Bio X Cell) and 2 µg/ml anti-CD28 mAb (Clone#37.51, Cat#BE0015-1, Bio X Cell), or 0.2 million/ml irradiated APCs and CBir1 peptide (ThermoFisher Scientific) in the presence or absence of acetate (10 mM, Sigma Aldrich), propionate (0.5 mM, Sigma Aldrich), or butyrate (0.5 mM, Sigma Aldrich), under neutral (without exogeneous cytokines), Th1 (10 ng/ml IL-12), Th17 (15 ng/ml TGFβ, 30 ng/ml IL-6, 10 µg/ml anti-IFNγ mAb, 5 µg/ml anti-IL-4 mAb), or Treg (5 ng/ml TGFβ and 10 µg/ml anti-IFNγ mAb) polarization conditions. Cells were cultured at 37 °C with 5% CO2. On day 5, the cell yield was about 2 million/ml.
3
Immunosuppressive Effect of MDSCs on T-Cell Proliferation
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MDSCs were isolated from PBMCs of HCs and PMN patients using a cell sorter (Influx, Becton Dickinson, Franklin Lakes, NJ, USA). CD4+ T cells (2 × 105 cells/well) were co-cultured in the absence or presence of autologous MDSCs at 1:1 ratio from HCs or PMN patients with 5 μg/mL plate-bound anti-CD3 mAb (BioXcell, West Lebanon, NH, USA) and 1 μg/mL soluble anti-CD28 mAb (BioLegend, San Diego, California, USA) in 96-well flat-bottom plate for 3 days, and T-cell proliferation was determined by measuring carboxyfluorescein diacetate succinimidyl ester (CFSE).
4
Treg Suppression Assay Protocol
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Purified Treg (CD4+YFP+, 25 × 103 cells/well) were cultured with or without whole splenocyte from CD3KO mice (7.5 × 104 cells/well), anti-CD3 mAb (0,05 μg/ml, BioXcell), TNF (50 ng/ml, Protein Service Facility, VIB, Belgium) and IL-2 (10 ng/ml, Peproteck) in a 96-well round plate in RPMI 1640–10% FCS. For suppression assays, after labeling with CellTrace Violet Proliferation Kit (Life technologies), Tconv cells (CD4+YFP−, 2.5 × 104 cells/well) were co-cultures with various Treg (CD4+YFP+) numbers and stimulated by splenocytes from CD3 KO mice (7.5 × 104 cells/well) and soluble anti-CD3 (0.05 μg/ml 2C11, BioXCell) in RPMI 1640–10% FCS.
5
Role of CD27 NK Cells in Th17 Induction
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Naı¨ve T cells (CD3 þ CD4 þ CD44 low CD62L high > 90% purity) were selected from the splenocytes of normal mouse using the Naive CD4 þ T cell Isolation Kit, mouse (Miltenyi Biotec). Naı¨ve T cells were activated by plate-bound anti-CD3 mAb (Bio X Cell, West Lebanon, NH, USA) and anti-CD28 mAb (Bio X Cell). To explore the role of CD27 NK cell subsets in the proliferation of T cells, 1 Â 10 4 CD27 high NK cells or CD27 low/À NK cells were co-cultured with carboxyfluorescein succinimidyl ester (CFSE)-labeled naı¨ve T cells for 3 d per well in 96-well plates in complete RPMI medium 1640 (NK: T ¼ 1:10). To determine the effects of CD27 NK cell subsets on Th17 expansion, IL-6 (40 ng/ml; eBioscience) and TGF-b (2 ng/ml; R&D Systems, Minneapolis, MN, USA) were added into the assay system. After co-culture, cells were collected for intracellular FCM, and the supernatants were used for ELISA detection of IL-17A (eBioscience).
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