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).
Cpg b odn 1826
Manufactured by InvivoGen
Sourced in United States
CpG B ODN 1826 is a synthetic oligodeoxynucleotide (ODN) that contains unmethylated CpG motifs. It functions 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
Hepes, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (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)
Cpg b odn2006, by InvivoGen (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)
Np 15 cgg, by Biosearch Technologies (2 mentions)
Sb203580, by Merck Group (1 mentions)
Jsh 23, by Merck Group (1 mentions)
Recombinant murine baff, by BioLegend (1 mentions)
Odn 20958, by Miltenyi Biotec (1 mentions)
8 protocols using cpg b odn 1826
1
Optimized Culture Conditions for Studying BAFF and APRIL
2
Murine Immune Cell Stimulation Reagents
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R848 (TLR7 ligand) and CpG-B ODN 1826 (murine TLR9 ligand) were purchased from Invivogen (San Diego, CA, USA). Lipopolysaccharide (LPS, TLR4 ligand from Salmonella Minnesota) was from Sigma-Aldrich(St. Louis, MO). Recombinant murine BAFF was from Biolegend (San Diego, CA). ODN2088 and ODN20958 were from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany). JSH23 (4-methyl-N1-(3-phenyl-propyl)-benzene-1,2-diamine, Sigma-Aldrich), SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole) (Sigma-Aldrich). Amino-actinomycin D (7-AAD) was obtained from BD Bioscience (San Jose, CA, USA). The non-secreting mouse myeloma cell line SP2/0 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The murine anti-U1-70 K (RNP) hybridoma cell line 2.73 was a gift of Dr. Sally Hoch [17 (link)]. Additional hybridomas included 172.4 (murine anti-CD100, ATCC), 11B11 (rat IgG1 anti-mouse IL-4, ATCC), Pab101 (murine IgG2a anti-SV40 large T antigen, ATCC), 111 (murine IgG1 anti-Ku), and 162 (murine IgG2a anti-Ku).
3
B cell TLR9-deficient mouse generation
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To generate B cell lineage-specific TLR9-deficient mice, lethally irradiated 7–8 weeks old C57BL/6 mice (CD45.1 haplotype) were reconstituted with 107 bone marrow cells of which 90% were from μMT mice and 10% were from either MyD88-deficient or WT mice. Mice were allowed to reconstitute for at least 8 weeks before immunization with both 100 μg NP(15)CGG (Biosearch Technologies) adsorbed into 100 μl Alum (ThermoFisher) in PBS and 65 μg CpG B (ODN 1826) (Invivogen) in PBS intraperitoneally.
4
Isolating and Culturing Lymphoid Cells
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For splenocyte and lymph node cell preparation, organs were mashed through a 70 μM cell strainer (BD Biosciences), as previously described (Rosser et al., 2014 (link)), and erythrocytes from spleens were lysed using Red Cell Lysis Buffer (Sigma-Aldrich). B cells were negatively purified by magnetic separation, according to manufacturer’s instructions (Miltenyi Biotec). Cells were cultured with either RPMI 1640 (Sigma-Aldrich) containing L-glutamine and NAHCO3 or Iscove’s Modified Dulbecco’s Medium (IMDM; Pan Biotech), enriched in AhR agonists (Veldhoen et al., 2009 (link)), supplemented with L-Glutamine and 25mM HEPES. Media were supplemented with 10% fetal calf serum (LabTech), 1% Penicillin/streptomycin (100U/ml Penicillin+100 μg/ml streptomycin; Sigma-Aldrich) and 50 μM 2-Mercaptoethanol (ThermoFisher Scientific). Cells were cultured at 37°C with 5% CO2.
Total lymphocytes, B cells and B cell subsets were cultured for 48h with CpGb ODN1826 (1 μM; Invivogen), LPS (1 μg/ml; Sigma-Aldrich) ± anti-mouse IgM (10 μg/ml; Jackson ImmunoResearch). or anti-CD40 (10 μg/ml; BioXcell). In addition, AhR agonist FICZ (100nM; Enzo LifeSciences) was added to culture. For 48h culture, anti-IgM ± FICZ were added 24h into culture.
Total lymphocytes, B cells and B cell subsets were cultured for 48h with CpGb ODN1826 (1 μM; Invivogen), LPS (1 μg/ml; Sigma-Aldrich) ± anti-mouse IgM (10 μg/ml; Jackson ImmunoResearch). or anti-CD40 (10 μg/ml; BioXcell). In addition, AhR agonist FICZ (100nM; Enzo LifeSciences) was added to culture. For 48h culture, anti-IgM ± FICZ were added 24h into culture.
5
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).
6
In vivo activation of DC subsets
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NOD/LtJ (NOD) mice were maintained at the UMS006 animal facility (Toulouse). C57Bl6/JRj (B6) mice were purchased from Janvier Labs (Le Genest Saint Isle, France).
For the in vivo activation of DC subsets, mice were i.v. injected with 5 μg of lipopolysaccharide (LPS) from Escherichia coli 0111:B4), 10 μg of CpG-B ODN1826, and 10 μg of Poly I:C (InvivoGen, Toulouse, France).
For in vivo FLT3L treatment, mice were injected subcutaneously (s.c.) with 5 × 106 B16-Flt3L melanoma cells (21 (link)).
All experiments involving animals were performed in accordance with national and European regulations and the Institut National de la Santé et de la Recherche Médicale institutional guidelines. Protocols were approved by the “Midi Pyrénées” ethical committee.
For the in vivo activation of DC subsets, mice were i.v. injected with 5 μg of lipopolysaccharide (LPS) from Escherichia coli 0111:B4), 10 μg of CpG-B ODN1826, and 10 μg of Poly I:C (InvivoGen, Toulouse, France).
For in vivo FLT3L treatment, mice were injected subcutaneously (s.c.) with 5 × 106 B16-Flt3L melanoma cells (21 (link)).
All experiments involving animals were performed in accordance with national and European regulations and the Institut National de la Santé et de la Recherche Médicale institutional guidelines. Protocols were approved by the “Midi Pyrénées” ethical committee.
7
B cell TLR9-deficient mouse generation
8
Herpes Simplex Virus-Induced Cytokine Detection
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Herpes simplex virus type 1 (HSV-1) strain 2931 (originally obtained from Dr. Carlos Lopez, then at the Memorial Sloan-Kettering Cancer Center, New York, USA) was grown and tittered in VERO cells. HSV-1 was used to stimulate PBMC or pDC at an MOI of 1 and Influenza A Virus (IAV) strain PR8 (Charles River Laboratories, Wilmington Massachusetts, USA) was used at an MOI of 2. For intracellular cytokine detection, 5 μg/ml of Brefeldin A (Sigma-Aldrich) was added 2 hours prior to the end of the incubation period. CpG-B ODN #1826 (Invivogen) was used at 5 μg/ml for stimulation in all experiments.
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