Cell migration assays were performed using Transwell chambers (pore size 5 μm, Costar, Corning 3421, Corning, NY, USA). THP-1 cells (2 × 106 in 200 μl) were re-suspended in serum-free RPMI 1640 medium and added to the upper chamber. Supernatant from infected 786-O cells was mixed with 1 μg/ml neutralizing antibody against human GM-CSF (502203, BVD2-23B6, Biolegend) or control IgG2a (400515, Rat IgG2a, Biolegend) and incubated for 30 min. Then, 600 μl of medium containing the supernatant was added to the lower chamber as the chemoattractant. After 3 h or 6 h of incubation at 37 °C in a 5% CO2 humidified atmosphere, the migrated cells in the lower chamber were counted.
Bvd2 23b6
Manufactured by BioLegend
Sourced in United States
The BVD2-23B6 is a monoclonal antibody product offered by BioLegend. It is designed for use in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Bvd2 21c11, by BioLegend (3 mentions)
Transwell chamber, by Corning (1 mentions)
Rat igg2a, by BioLegend (1 mentions)
Sulfatide, by Merck Group (1 mentions)
Mp4 25d2, by BioLegend (1 mentions)
α galactosylceramide α galcer, by Avanti Polar Lipids (1 mentions)
Horseradish peroxidase conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
O phenylenediamine dihydrochloride substrate, by Merck Group (1 mentions)
Uquant microplate reader, by Agilent Technologies (1 mentions)
4 protocols using bvd2 23b6
1
Transwell Assay for Cell Migration
2
Lipid Stimulation of Monocyte-Derived Dendritic Cells
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Monocytes, Mo-DCs or CD1-transfected C1R cells were cultured with sulfatide (30–0.04 μg/mL, Sigma-Aldrich), GM1 (50–0.07 μg/mL, Sigma-Aldrich), α-Galactosylceramide (α-GalCer) (at 50 ng/mL or 50–3.12 ng/mL, Avanti polar lipids, Alabaster, AL, USA) or α-Gal-(1-2)-αGalCer (300–50 ng/mL). Lipids, with the exception of α-GalCer, were first dissolved in methanol or PBS 0.5% Tween 20 and then diluted in non-supplemented RPMI to have a maximum of 1% vehicle in culture. α-GalCer was resuspended in PBS and directly diluted in non-supplemented RPMI. After 4 h, an iNKT cell line or T cell clones were added and 40 h later, supernatants were collected for cytokine production determination by ELISA. The following antibody pairs from Biolegend were used: purified anti-human GM-CSF (BVD2-23B6) and biotinylated anti-human GM-CSF (BVD2-21C11); purified anti-human IL-4 (8D4-8) and biotinylated anti-human IL-4 (MP4-25D2).
3
Cytokine Quantification by ELISA
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Levels of GM-CSF, IFN-γ, IL-4 and IL-10 on culture supernatants were assessed by ELISA. For IFN-γ, IL-4 and IL-10, ELISA kits from BioLegend San Diego, CA, USA were used according to the manufacturer’s instructions. GM-CSF concentration in the supernatants was measured using a purified anti-GM-CSF mAb (BVD2-23B6, BioLegend, San Diego, CA, USA) as capture antibody and a biotinylated anti-GM-CSF mAb (BVD2-21C11, BioLegend, San Diego, CA, USA) as detection antibody. Signal was detected by incubating plates with horseradish peroxidase-conjugated streptavidin (Invitrogen, Eugene, Oregon, USA), followed by o-phenylenediamine dihydrochloride (OPD) substrate (Sigma, St. Louis, MO, USA). Absorbances were read using a Bio-Tek (Winooski, Vermont, USA) uQuant microplate reader and the Gen5 software.
4
Invariant NKT Cell Activation Assays
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Invariant natural killer T cell activation assays were performed using CD1d-transfected C1R cells as APCs or soluble mouse CD1d coated in 384-well plates.
For assays using APC, CD1d-transfected C1R cells were cultured for 4 h with 5 or 25 ng/ml of α-GalCer alone or together with increasing doses (1, 10, 20×) of L. infantum Exo, EV, or VDE. The human polyclonal primary iNKT cell line was then added and supernatants from this co-culture were harvest 40 h later and assayed for GM-CSF by ELISA using purified (BVD2-23B6) and biotinylated (BVD2-21C11) anti-GM-CSF mAbs (Biolegend). CD1d (NIH tetramer core facility) was immobilized on 384-well Maxisorp treated microplates (Nunc, Rochester, NY, USA). After overnight incubation at 37°C, a mixture of 25 or 100 ng/ml α-GalCer and L. infantum Exo, EV, VDE, or hEV was added for 24 h. In some experiments, α-GalCer (25 ng/ml) was added 8 h before or after Exo, VDE, or EV (20×). After extensive washing, the 24.8 iNKT cell hybridoma (25,000/well) was added for further 20 h. Culture supernatants were recovered, and IL-2 concentrations were determined by ELISA using purified (JES6-1A12) and biotinylated (JES6-5H4) anti-IL-2 mAbs (Biolegend).
For assays using APC, CD1d-transfected C1R cells were cultured for 4 h with 5 or 25 ng/ml of α-GalCer alone or together with increasing doses (1, 10, 20×) of L. infantum Exo, EV, or VDE. The human polyclonal primary iNKT cell line was then added and supernatants from this co-culture were harvest 40 h later and assayed for GM-CSF by ELISA using purified (BVD2-23B6) and biotinylated (BVD2-21C11) anti-GM-CSF mAbs (Biolegend). CD1d (NIH tetramer core facility) was immobilized on 384-well Maxisorp treated microplates (Nunc, Rochester, NY, USA). After overnight incubation at 37°C, a mixture of 25 or 100 ng/ml α-GalCer and L. infantum Exo, EV, VDE, or hEV was added for 24 h. In some experiments, α-GalCer (25 ng/ml) was added 8 h before or after Exo, VDE, or EV (20×). After extensive washing, the 24.8 iNKT cell hybridoma (25,000/well) was added for further 20 h. Culture supernatants were recovered, and IL-2 concentrations were determined by ELISA using purified (JES6-1A12) and biotinylated (JES6-5H4) anti-IL-2 mAbs (Biolegend).
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