For B/T cells co-cultures, naïve or memory B cells were co-cultured with CD4+ T cells (2 × 104 B cells - 2 × 104 CD4 T cells) in the presence of endotoxin-reduced SEB (500 ng/ml; Sigma Aldrich) in cRPMI supplemented with 10% heat-inactivated FBS. Where described, anti-IL10 (10 μg/ml - clone JES3–9D7 - Biolegend LEAF™ purified antibody), anti-SUCNR1/GPR91 (20 μg/ml – Novus Biological), anti- IL21R (10 μg/ml - clone 17A12 - Biolegend LEAF™ purified antibody) or succinate (2 mM – Sigma-Aldrich) were added during the co-cultures. Sodium azide and other preservatives were removed from the antibody preparations by protein desalting with Zeba Spin Desalting Columns (7K MWCO; Thermo Fisher). IgM and IgG concentrations were measured at day 12 in the corresponding supernatants with Flex Set Kit (BD Biosciences). For T cell-independent B cell differentiation, naïve B cells (5 × 104 cells) were co-cultured with irradiated (77Gy) human-CD40L transfected fibroblasts43 (link) (0.5 × 104 cells) in cRPMI supplemented with 10% heat-inactivated FBS. Recombinant human IL10 (Biolegend, 500 ng/ml) and/or succinate (2 mM – Sigma-Aldrich) were added during the co-cultures. IgG and IgM concentrations were measured at day 12 in the corresponding supernatants with Flex Set Kit (BD Biosciences).
Flex set kit
Manufactured by BD
Sourced in United States
The Flex Set Kit is a lab equipment product designed for versatile sample preparation. It includes a set of modular components that can be configured to accommodate various laboratory tasks. The kit provides a flexible and adaptable solution for sample handling and processing needs in the lab environment.
Automatically generated - may contain errors
Lab products found in correlation
Anti il 10, by BioLegend (2 mentions)
Succinate, by Merck Group (2 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (2 mentions)
Anti sucnr1 gpr91, by Novus Biologicals (2 mentions)
Recombinant human il 10, by BioLegend (2 mentions)
Annexin 5 apoptosis detection kit, by BD (2 mentions)
Recombinant human il3, by BD (2 mentions)
Facsaria 3 flow cytometer, by BD (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Infinite m200, by Tecan (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Fcap array software, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Cellquest pro software, by BD (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
9 protocols using flex set kit
1
B/T Cell Co-culture Assay for Immunoglobulin Production
2
B/T Cell Co-culture Assay for Immunoglobulin Production
3
Cytokine and Phenotypic Analysis of Plasmacytoid Dendritic Cells
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PDCs were cultured as described in the paragraph above, and cytokine levels in the corresponding supernatants were measured with Flex Set Kit (BD Biosciences). For flow cytometry analysis, cells were stained with anti-CD80 APC (Clone L307), anti-CD86 PE (Clone FUN-1), anti-CD83 FITC (Clone HB15e), anti-CD40 PE (Clone 5C3), anti-HLADR APC-H7 (Clone G46–6), anti-CD123 PE (Clone 9F5), anti-CXCR4 PECy7 (Clone 12G5), anti-CXCR3 Brilliant Violet 785 (Clone GO25H7) or anti-CCR7 APC (Clone 3D12). To assess the effect of IL3 on cell viability, activated pDCs were treated with recombinant human IL3 (50 ng/ml – BD Biosciences), cultured for an additional 24 h and then stained with the annexin V–apoptosis detection kit (BD Biosciences) following the manufacturer’s instructions.
4
Cytokine Profile in Mouse Spleen
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Levels of IL-1β, IL-6, IL-17A, IFN-γ, TNF, and MCP-1 in the serum and spleen of mice were evaluated by Cytometric Bead Array (CBA) with Flex Set kit from BD Biosciences according to the manufacturer’s instructions using a FACSAria III flow cytometer (Becton, Dickinson and Company, USA). Protein in the spleens was extracted by tissue homogenate method and the concentrations of that were measured by BCA (Thermo, Rockford, USA).
5
Cytokine and Phenotypic Analysis of Plasmacytoid Dendritic Cells
6
Quantifying Serum Cytokines and IgE Levels
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Levels of IL 4, IL-5, and IL-13 in serum were measured using an enhanced sensitivity cytometric bead array (CBA, Flex Set Kits; BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer’s guidelines. IgE in serum was measured by ELISA. Briefly, 96-well high-binding ELISA plates (Greiner, Sigma-Aldrich, Deisenhofen, Germany) were coated with monoclonal anti-IgE antibodies (clone R35-72, BD Biosciences, Heidelberg, Germany) overnight. Serum samples were diluted in 1% (w/v) BSA in PBS/0.05% (v/v) Tween 20 and incubated overnight at 4°C. Afterward, plates were incubated with anti-IgE conjugated with HRP (clone 23G3, Southern Biotech, Birmingham, AL, USA) for 3 h at room temperature. For the colorimetric detection, TMB (Sigma) was used as a substrate. Absorbance was measured at 450 nm in ELISA reader (Infinite M200, Tecan) and IgE concentrations calculated according to standard curve.
7
Blackcurrant Leaf Extract Modulates eNOS Phosphorylation in HUVECs
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HUVEC (1 × 106) were placed in a 75 cm2 culture bottles and after 24 h, the cells were incubated with 2.5 or 15 μg GAE/ml blackcurrant leaf extract for further 24 h. After the incubation, the cells were harvested by trypsinization and centrifuged (100×g, 6 min, RT). The obtained cell pellets were subsequently denatured with denaturation buffer previously diluted in deionised water and supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail 2 (5 min, 100 °C). The presence of DNA in denaturated samples were remedied by adding DNase I (15 min, 37 °C) which was further inactivated by sample heating at 75 °C for 10 min. The obtained cells lysates were stored at −70 °C until determination of protein concentration by BCA assay or eNOS phosphorylation. Before further analysis, the samples were centrifuged to pellet the debris (10,000×g, 3 min). The measurements of endothelial nitric oxide synthase (eNOS) phosphorylation at Ser-1177 and Thr-495 were performed with the use of the commercial BD™ Flex Set Kits, for Phospho eNOS S1177 and Phospho eNOS T495 according to the manufacturer’s instructions. The samples were measured on a Becton Dickinson FACSCanto II flow cytometer and the results were analysed with Becton Dickinson FCAP Array software.
8
Cytokine and TNF-α Profiling in BALF
9
Measuring Vγ2Vδ2 T Cell Cytokines
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To measure the cytokines released from Vγ2Vδ2 T cells, the supernatants were harvested from the samples co-cultured with the T cells and tumor cells. Flex Set kits (BD, San Jose, USA) were used to measure the human IFNγ, TNFα, and Granzyme B according to the manufacturer’s instructions. To determine the production of cytokines induced by the antibodies, the raw values were subtracted from the values of E+T groups in the absence of the tested antibodies.
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