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).
Bvd2 21c11
Manufactured by BioLegend
Sourced in United States
The BVD2-21C11 is a mouse monoclonal antibody that recognizes the B7-2 (CD86) protein. B7-2 is a co-stimulatory molecule expressed on antigen-presenting cells that provides a secondary signal required for the activation of T cells.
Automatically generated - may contain errors
Lab products found in correlation
Bvd2 23b6, by BioLegend (3 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)
Golgiplug, by BD (1 mentions)
Golgistop, by BD (1 mentions)
Fastimmune, by BD (1 mentions)
Tnfα alexa 488, by BioLegend (1 mentions)
Ifnγ alexa647, by BioLegend (1 mentions)
Gm csf pe, by BioLegend (1 mentions)
Il 2 bv421, by BioLegend (1 mentions)
4 protocols using bvd2 21c11
1
Lipid Stimulation of Monocyte-Derived Dendritic Cells
2
Cytokine Quantification by ELISA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
3
Myelin-Specific CD8+ T Cell Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
T2 cells (1 × 105 cells per condition) expressing HLA-A3 and/or HLA-A2 were pulsed overnight with 10 µg/mL of the indicated myelin peptide or no antigen. Myelin tetramer-positive CD8+ T cells (5 × 104 to 2 × 105) were added to the pulsed T2 cells (total volume, 100 µL) and stimulated for 6 h in the presence of 1:500 GolgiStop (BD), 1:500 GolgiPlug (BD), and 1:200 CD28/CD49d (FastImmune; BD). Cells were washed with FACS buffer and stained with the cell surface antibodies as above (anti-CD8, dump channel antibody mixture, and live/dead dye). Cells were washed, fixed, and stained with antibodies for intracellular cytokines in permeabilization buffer. Intracellular antibodies included IFN-γ Alexa 647 (BioLegend; 4S.B3), TNFα Alexa 488 (BioLegend; Mab11), IL-2 BV421 (BioLegend; MQ1-17H12), GM-CSF PE (BioLegend; BVD2-21C11), CD107a APC/Cy7. Cells were then washed and collected on an LSRFortessa.
4
Invariant NKT Cell Activation Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!