Fluorescence-activated cell sorting analysis was performed using a BD LSRFortessa™ (BD Biosciences, San Jose, CA). The following antibodies were used to classify cells: anti-CD19-fluorescein isothiocyanate (FITC) (Miltenyi Biotec, Friedrich Gladbach, Germany), anti-CD3-allophycocyanin (APC), anti-CD4-APC, anti-CD4-phycoerythrin (PE), anti-CD24-peridinin chlorophyll protein complex-Cy 5.5 (PerCP Cy5.5), anti-CD25-APC, anti-CD27-PE, anti-CD39- brilliant violet 421 (BV421), anti-CD45-V500, anti-CD73-APC, anti-IFN-γ-BV421, and anti-IL-10-BV421 (all from BD PharMingen, Franklin Lakes, NJ), anti-CD80-APC, anti-CD86-APC monoclonal antibody (mAb) (Biolegend, San Diego, CA), Annexin V- FITC (BD PharMingen), and DAPI (Cell Biolabs, San Diego, CA). The CD19+CD24hiCD27+ B cell subset was sorted using a Moflo XDP cell sorter (Beckman Coulter, Brea, CA) with 90% to 95% purities.
Anti il 10 bv421
Manufactured by BD
Sourced in United States
Anti-IL-10-BV421 is a fluorochrome-conjugated antibody that binds to the interleukin-10 (IL-10) cytokine. It is designed for use in flow cytometry applications to detect and analyze IL-10 expression in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin, by Merck Group (2 mentions)
Annexin 5 fitc, by BD (1 mentions)
Anti cd45 v500, by BD (1 mentions)
Anti cd4 apc, by BD (1 mentions)
Anti cd3 allophycocyanin, by BD (1 mentions)
Anti cd27 pe, by BD (1 mentions)
Anti cd80 apc, by BioLegend (1 mentions)
Anti cd25 apc, by BD (1 mentions)
Anti cd4 phycoerythrin, by BD (1 mentions)
Moflo xdp cell sorter, by Beckman Coulter (1 mentions)
Lsrfortessa, by BD (1 mentions)
Anti ifn γ bv421, by BD (1 mentions)
Anti cd73 apc, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Cell Biolabs (1 mentions)
Anti cd19 fluorescein isothiocyanate fitc, by Miltenyi Biotec (1 mentions)
Facsaria 2 flow cytometer, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Cd38 fitc, by BD (1 mentions)
Cd24 pe, by BD (1 mentions)
Cd19 pe cy7, by BD (1 mentions)
3 protocols using anti il 10 bv421
1
Immunophenotyping of B Cell Subsets
2
Cytokine Expression in Acute Hepatitis E
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The expression of cytokines IL-10 and TGF-β by B cells and its subsets were assessed by intracellular cytokine staining. PBMCs from 36 acute hepatitis E patients, 29 hepatitis E recovered individuals and 51 healthy controls were incubated with 1 μM CpG-B ODN2006 (InvivoGen, CA, USA) for 72 h at 37°C. Phorbol myristate acetate (PMA) (25 ng/mL) and ionomycin (1 μg/mL) (Sigma, USA) were added in the last 5 h in the presence of 10 μg/mL Brefeldin-A (Sigma, USA). Alternatively, PBMCs were stimulated with HEV-rORF2p (10 μg/mL) for 72 h. Cells were then surface stained for the markers CD19 PE-Cy7, CD24 PE and CD38 FITC (BD Biosciences, CA, USA), fixed, permeabilized, and stained intracellularly with anti-IL-10 BV421 and TGF-β PerCP-Cy5.5 antibodies (BD Biosciences, CA, USA). Isotype and Fluorescence Minus One (FMO) controls were used in all sets of experiments. For each sample, 50,000 events were acquired in BD FACS Aria-II flow cytometer and data were analyzed using BD FACS Diva software (BD Biosciences, CA, USA). Data from stimulated cells were analyzed after normalization with unstimulated cells. The gating strategy is depicted in (Supplementary Figure S2 ).
3
Phenotypic Analysis of Activated T Cells
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Cells (10 7 /ml) were plated and incubated at 37°C in an atmosphere of 5% CO 2 for 5 h in RPMI-1640 (Sigma-Aldrich, Steinheim, Germany) with penicillin-streptomycin (Sigma-Aldrich), L-glutamine (Sigma-Aldrich), and foetal bovine serum (Sigma-Aldrich) in the presence of 50 ng/ml phorbol 12-myrisate 13-acetate (Sigma-Aldrich), 250 ng/ml ionomycin (Sigma-Aldrich), and 2 μg/ml monensin (Sigma-Aldrich). Thereafter, the cells were immediately stained with the following antibodies from BD Biosciences: anti-CD3-BV650 (563852), anti-CD4-AF700 (557922), and anti-CD8-APC-H7 (560179). Next, they were fixed and permeabilized according to the manufacturer's protocol (Permeabilization Buffer 00-8333-56, Fixation/Permeabilization Diluent 00-5223-56, and Fixation/Permeabilization Concentrate 00-5123-43; eBioscience, San Diego, CA, USA). Finally, the cells were stained with the following antibodies: anti-IL5-PE (554395; BD Biosciences), anti-IL10-BV421 (564053; BD Biosciences), anti-IL17-BV510 (563295; BD Biosciences), anti-IL22-PerCP eFluor (710 46-7229-42; eBioscience), and anti-interferon (IFN)-γ-BV488 (557718; BD Biosciences). Brilliant Stain Buffer was added to the antibody cocktail.
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