Peripheral blood mononuclear cells (PBMCs) were isolated using lymphocyte separation medium (Ficoll-Hypaque density gradient, Axis-shield, Germany) from 10 ml fresh heparinized blood sample. And then PBMCs were incubated with 1 μM CpG-B ODN2006 (In vivoGen, San Diego, CA, USA) for 96 h at 37°C. PMA (3 ng/ml) and ionomycin (100 ng/ml) were added during the last 4h in the presence of 10 μg/ml brefeldin A (all from Sigma, St Louis, MO, USA). Cells were then surface stained for the markers CD19 Pacific Blue-A, and stained intracellularly with anti-IL-10 APC. So we could assess the ability and the frequency of IL-10 production from purified B cells (14 (link)). All antibodies including CD19-PE and IL-10-APC used in flow cytometry were ordered from BD Pharmingen (BD Pharmingen, San Diego, CA, USA). PBMCs were acquired on a FACS Canton flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). FlowJo 7.6.1 (Tree Star Inc., Ashland, OR, USA) was used as the flow analysis software.
Cpg b odn2006
Manufactured by InvivoGen
Sourced in United States
CpG-B ODN2006 is a synthetic oligodeoxynucleotide (ODN) that contains unmethylated CpG motifs. It acts as a toll-like receptor 9 (TLR9) agonist, which can stimulate the immune system by activating B cells and plasmacytoid dendritic cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal calf serum (fcs), by Thermo Fisher Scientific (3 mentions)
Brefeldin a, by Merck Group (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Cpg b odn 1826, by InvivoGen (2 mentions)
Mouse baff recombinant protein, by R&D Systems (2 mentions)
Human baff recombinant protein, by R&D Systems (2 mentions)
Mouse april recombinant protein, by R&D Systems (2 mentions)
Human april protein, by R&D Systems (2 mentions)
Anti mouse il 10 af 417 na, by R&D Systems (2 mentions)
Anti mouse taci af1041, by R&D Systems (2 mentions)
Anti human bcma af193, by R&D Systems (2 mentions)
24 wells cell culture plates, by BD (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Ficoll hypaque density gradient, by Axis-Shield (1 mentions)
Cd19 pe, by BD (1 mentions)
Il 10 apc, by BD (1 mentions)
Celltrace violet ctv cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
8 protocols using cpg b odn2006
1
Assessing IL-10 Production in B Cells
2
Optimized Culture Conditions for Studying BAFF and APRIL
3
B Cell Proliferation Assay with CD38 Inhibitors
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Isolated PB B cells were labelled using the CellTrace Violet (CTV) Cell Proliferation Kit (Life Technologies), according to manufacturer’s instructions, and cell proliferation was measured by CTV dilution. The cells were cultured in 96-well plates at 1 × 104 cells/well in complete medium (Table S5 ) and stimulated with 2.5 µg/ml goat α-human IgA/IgG/IgM F(ab′)2 fragments (α-Ig; Jackson ImmunoResearch Laboratories) in combination with 20 ng/ml human recombinant IL-2 (R&D Systems) and 2.5 µg/ml TLR9 agonist, CpG-B ODN 2006 (InvivoGen) for 5/7 d with or without antibodies recognizing CD38: α-CD38 mouse clone HB-7 (Ultra-LEAF Purified; Biolegend) and daratumumab (trade name Darzalex, human, IgG1κ, HuMax-CD38, Genmab/Janssen) both at a concentration of 1 µg/ml (de Weers et al., 2011 (link); Laubach et al., 2014 (link); Matas-Céspedes et al., 2017 (link)). The proliferation was assessed as expansion index. Data were acquired by flow cytometry.
4
Optimized Culture Conditions for Studying BAFF and APRIL
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RPMI and DMEM media (Invitrogen, Carlsbad, CA, USA) were used for all mouse and human cultures, respectively. Media were supplemented with 10% FCS, L-glutamine, 2ME, HEPES and Pen/Strep; all from Invitrogen (Carlsbad, CA, USA). Stimulatory factors were used at the following concentrations: LPS, 30ug/ml; mouse BAFF recombinant protein, 50ng/ml; human BAFF recombinant protein, 50ng/ml; mouse APRIL recombinant protein, 50 ng/ml; human APRIL protein, 50ng/ml; all from R&D Systems (Minneapolis, MN, USA). CpG B ODN 1826, 1μM; CpG B ODN 2006, 1μM; all purchased from Invivogen (San Diego, CA, USA). CpG stimulations were conducted from 5-48 hrs. In vitro neutralization of protein ligands BAFF and IL-10 was achieved with purified anti-human BAFF (AF124), anti-mouse BAFF (AF2106), anti-human IL-10 (AF-217-NA) and anti-mouse IL-10 (AF-417-NA) all from R&D Systems (Minneapolis, MN, USA) and used at 50μg/ml. In vitro neutralization of BAFF receptors TACI, BAFF-R and BCMA was achieved with purified anti-mouse TACI (AF1041), anti-mouse BAFF-R (AF1357), anti-mouse BCMA (AF593) and anti-human TACI (AF174), anti-human BAFF-R (AF1162) and anti-human BCMA (AF193) all from R&D Systems (Minneapolis, MN, USA) and used at 1μg/ml. Cells were cultured in 96-, 48-, or 24-wells cell culture plates (BD, San Diego, CA, USA).
5
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 ).
6
Enumerating Env-specific Plasma Cells and Memory B Cells
7
Differentiation of CAL-1 Cells into GM-CSF-Induced Dendritic Cells
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CAL-1 cells, obtained under a material transfer agreement, were cultured in complete growth medium RPMI 1640 containing 2mM L-alanyl-L-glutamine (Pan Biotech Ref. P04-18500, Chemie Brunschwig, Basel, Switzerland), supplemented with 10% FCS (Gibco Ref. 10270-106; LuBioScience, Luzern, Switzerland) and 1% penicillin/streptomycin (BioWhittaker Lonza; VWR Scientific, Nyon, Switzerland) in suspension tissue culture flasks or plates (Greiner Bio-One; Huberlab, Aesch, Switzerland). Floating cells were passaged to new culture flasks for cell maintenance. Where indicated, CAL-1 cells (2x 105 cells/ml) were exposed to 10ng/ml human GM-CSF (Peprotech, LuBioScience; catalog #300-03) for 3 days, referred to as GM/CAL-1 cells, washed, resuspended at a density of 1x 106 cells/ml in complete growth medium without GM-CSF and then left untreated or matured for another 18-19h with either the TLR7/8 agonist Resiquimod R848 (10μg/ml; Sigma–Aldrich, Buchs, Switzerland; #SML0196) or the TLR9 ligand CpG-B ODN 2006 (1μM; InvivoGen; LabForce, Muttenz, Switzerland; #tlrl-2006-1).
8
Culturing Dendritic Cells and Plasmacytoid Dendritic Cells
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DC and pDC were cultured in RPMI 1640 Medium GlutaMAX (Life Technologies) containing 10% Fetal Calf Serum (Hyclone), 100 U/ml Penicillin/Streptomycin (Gibco), MEM Non-Essential Amino Acids (Gibco), and 1 mM NA pyruvate (GIBCO). DCs were cultured at 106/ml in flat bottom plates for 24 h in the presence of 50 ng/ml rhTSLP- where not differently specified (R&D Systems) or 100 ng/ml ultrapure LPS (InvivoGen).
pDCs were cultured at 106/ml in flat-bottom plates for 24 h in the presence of 15 µg/ml CpGB ODN 2006 (InvivoGen).
pDCs were cultured at 106/ml in flat-bottom plates for 24 h in the presence of 15 µg/ml CpGB ODN 2006 (InvivoGen).
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