PBMCs were cultured in 96-well flat-bottom plates in Basal Medium Eagle (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin (all from Thermo Fisher Scientific). PBMCs were stimulated with recombinant Human IL-3 (100 ng/mL; PEPROTECH, Rocky Hill, NJ, USA) and a TLR7 agonist, imiquimod (R837) (100 ng/mL; InvivoGen, San Diego, CA, USA) or a TLR9 agonist, CpG ODN 2216 (5 μg/mL; Miltenyi Biotec, Bergisch Gladbach, Germany) for 6 h at 37 °C in a 5% CO2 incubator. GolgiPlug (100 ng/mL; BD Biosciences) was added during the final 3 h of stimulation to block cytokine secretion. After staining the cell-surface antigens, intracellular cytokines were stained using the BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences), anti-IFN-α-APC (Miltenyi Biotec), and anti-tumor necrosis factor α (TNF-α)-PE-Cy7 (BD Biosciences), or their isotype control antibodies.
Cpg odn 2216
Manufactured by Miltenyi Biotec
Sourced in United States
CpG ODN 2216 is a synthetic oligodeoxynucleotide containing unmethylated CpG motifs. It acts as a toll-like receptor 9 (TLR9) agonist, which can stimulate the innate immune system.
Automatically generated - may contain errors
Lab products found in correlation
Human il 3, by Miltenyi Biotec (2 mentions)
Brefeldin a, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Basal medium eagle, by Thermo Fisher Scientific (1 mentions)
Imiquimod r837, by InvivoGen (1 mentions)
Recombinant human il 3, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm fixation permeabilization solution kit, by BD (1 mentions)
Tnf α pe cy7, by BD (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Golgiplug, by BD (1 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
Imiquimod imq, by InvivoGen (1 mentions)
Resiquimod r848, by InvivoGen (1 mentions)
Rpmi 1640 medium, by Harvard Bioscience (1 mentions)
Legendplex kit, by BioLegend (1 mentions)
Poly 1 c, by InvivoGen (1 mentions)
Hepes buffer solution, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
4 protocols using cpg odn 2216
1
Cytokine Profiling of Activated PBMCs
2
Stimulation and Analysis of PBMCs
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Peripheral blood mononuclear cells (PBMCs) were obtained from HD and MM patients by Ficoll gradient. PBMCs (1 × 106 cells/mL) were cultured in RPMI 1640 medium (Biochrom GmbH, Berlin, Germany) with 10% fetal bovine serum (FBS) (Biochrom GmbH, Holliston, MA, USA) and 20 ng/mL human IL-3 (Miltenyi Biotec, Bergisch Gladbach, Germany). Total PBMCs (1 × 106 cells/mL) were stimulated with resiquimod (R848) or imiquimod (IMQ) 5 μg/mL (Invivogen, San Diego, CA, USA) and CpG-ODN 2216 6 μg/mL (Miltenyi Biotec, Bergisch Gladbach, Germany) for 2 h and 6 h, respectively. Brefeldin A (1 μg/mL; Sigma-Aldrich, St. Louis, MO, USA) was added after 1 h or 4 h (in samples stimulated for 2 h or 6 h, respectively).
3
Cytokine Production by DCs and Monocytes
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Sorted DCs and monocytes were cultured in RPMI 1640 medium, supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 10 mM HEPES buffer solution (all obtained from Invitrogen), and 50 µM 2-mercaptoethanol (Sigma-Aldrich) at a density of 50,000 per 50 μl in 96 well V-bottom plates. Cells were stimulated with 10 ng/ml LPS (Sigma Aldrich), 1 μg/ml R848 (Invivogen), 25 μg/ml poly I:C (Invivogen), or 1 µM CpG ODN 2216 (Miltenyi Biotec) alone or in various combinations for 20 h at 37oC, 5% CO2. Supernatants were harvested and cytokines measured using ELISA kits for IL-1β, IL-23, IFN-λ (R&D Systems), IL-6, IL-12p70 (BD Biosciences), and IFN-α (Interferon Source) or by flow cytometry using a LegendPlex kit (Biolegend) that was acquired on an LSR Fortessa X20 (BD Biosciences) and analyzed using VigeneTech software Version 7.0.
4
Isolation and Stimulation of Peripheral Blood pDCs
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Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats of Healthy Donors (HD) (courtesy of the Centro Trasfusionale, ASST Spedali Civili, Brescia) by Ficoll gradient. Peripheral blood pDCs were magnetically sorted with the Plasmacytoid Dendritic Cell Isolation Kit II (Miltenyi Biotec; cat. No. 130-097-415) and the pDCs purity was greater than 92% for all experiments and greater than 95% for bulk RNA-Sequencing experiments. Blood pDCs (5 x 105 cells/mL) were cultured for 24 hours in RPMI 1640 (Gibco; cat. No. 31870-025) with 10% FBS (Biospa; cat. No. S1810-500), Glutamine and 1% penicillin-streptomycin (Gibco; cat. No. 15070-063) or with the SN-mel, with the addition of human IL-3 (20 ng/mL; Miltenyi Biotec; cat. No. 130-095-071). pDCs were stimulated with R848 (5 μg/mL; Invivogen; cat. No. tlrl-r848-5), CpG-ODN 2216 (6 μg/mL; Miltenyi Biotec; cat. No. 130-100-244) or ADU-S100 (50 μg/mL; Invivogen; cat. No. TLRL-NACDA2R). Brefeldin A (1 μg/mL; Sigma; cat. No. B7651) was added after stimulation to block protein secretion and evaluate intracellular cytokines.
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