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 cd28 mab
Manufactured by BioXCell
Sourced in United States
Anti-CD28 mAb is a monoclonal antibody that targets the CD28 receptor on the surface of T cells. It is a laboratory reagent used for in vitro research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd3 mab, by BioXCell (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 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)
Anti cd3, by BioXCell (1 mentions)
Anti ahr, by Enzo Life Sciences (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Mini protean tgx stain free gel, by Bio-Rad (1 mentions)
Mouse anti human cd3 clone ucht1, by BD (1 mentions)
Alexa fluor 647 conjugated goat anti mouse igg, by Jackson ImmunoResearch (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 cd28 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
Activation of CD4+ T Cells under Treg Conditions
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CD4+ T cells were activated with anti-CD3 (Bio X Cell) and anti-CD28 mAb (Bio X Cell) in the presence or absence of glucose (7000 μg/ml) under Treg polarization conditions. Cells were collected on day 3. Proteins were extracted using a radio-immunoprecipitation buffer containing protease inhibitor cocktail, phosphatase inhibitor cocktail, and PMSF. Protein concentrations were determined using a Pierce BCA Protein Assay Kit (ThermoFisher Scientific). 10 μg protein of each sample was loaded into the Mini-PROTEAN TGX Stain-free Gels (BIO-RAD) and separated electrophoretically. Proteins were then transferred to a PVDF membrane. After blocking with 5% non-fat milk, the membrane was incubated with primary antibodies overnight at 4°C and then with secondary antibodies (1:5000) for 1 h at room temperature. After incubating with the substrate, blots were detected using the ChemiDocTM Imaging System. Primary antibodies: anti-AhR, 1:2000, Enzo Life Sciences; anti-β-actin, 1:2000, Cell Signaling Technology.
4
In vivo Anti-CD3 mAb Experiments
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In vivo anti-CD3 mAbs UCHT1 (mouse anti-human, isotype IgG1, catalog #BE0231), OKT3 (mouse anti-human, isotype IgG2a, catalog #BE0001-2), anti-CD28 mAb (mouse anti-human, isotype IgG2a, clone 9.3, catalog #BE0248), and in vivo mouse IgG1 isotype control (clone MOPC-21, catalog #BE0083) were obtained from BioXCell (West Lebanon, NH, USA) and used for LIGS experiments. Alexa Fluor 647-conjugated goat anti-mouse IgG (catalog #115-605-062) was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Allophycocyanin (APC)-conjugated mAbs, Mouse anti-Human TCRαβ (BD PharMingen, 563826), Mouse anti-Human CD3 (clone UCHT1, BD PharMingen, 555335), Mouse anti-Human CD3 (clone OKT3, eBioscience, 17-0037-41), and Mouse anti-Human CD28 (eBioscience, 17-0289-41) were used for routine flow cytometry analysis.
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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