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Cpg 1668

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CpG 1668 is a synthetic oligodeoxynucleotide (ODN) that contains an unmethylated CpG motif. CpG 1668 acts as a toll-like receptor 9 (TLR9) agonist, which can stimulate the innate immune response.

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Lab products found in correlation

Anti mouse igm f ab 2, by Jackson ImmunoResearch (1 mentions) Anti b220 apc, by Thermo Fisher Scientific (1 mentions) Pe donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions) Cpg 2216, by InvivoGen (1 mentions) Escherichia coli lps, by Merck Group (1 mentions) Golgiplug, by BD (1 mentions) Cellbind plate, by Corning (1 mentions) Lipopolysaccharides (lps), by InvivoGen (1 mentions) Kta fplc system, by GE Healthcare (1 mentions) Dq green bsa, by Thermo Fisher Scientific (1 mentions)

4 protocols using cpg 1668

1

Stimulation of Lymph Node B Cells

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Single cell suspensions were generated from lymph nodes of 8–10 week old mice, resuspended at 2×107 cells/ml, rested in serum free media at 37°C for 1 hour, and stimulated in 96 well V-bottom plates at 37°C with vehicle, CpG 1668 (Invivogen) or F(ab’)2 anti-mouse IgM (Jackson Immunoresearch) for the times indicated in the figure legend. Cells were fixed with pre-warmed 1% paraformaldehyde, washed, permeabilized with ice cold 100% methanol, washed, rehydrated in PBS, stained with antibodies against pERK or IκBα (Cell Signaling Technologies), washed, stained with PE-donkey anti-rabbit IgG (Jackson Immunoresearch) and anti-B220-APC (eBiosciences), and analyzed as described above.
Mills R.E., Lam V.C., Tan A., Cresalia N., Oksenberg N., Zikherman J., Anderson M., Weiss A, & Hermiston M.L. (2015). Unbiased modifier screen reveals that signal strength determines the regulatory role murine TLR9 plays in autoantibody production. Journal of immunology (Baltimore, Md. : 1950), 194(8), 3675-3686.
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2

Cytokine Production by ILCs and APCs

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ILCs and APC populations were sorted on a FACSAria and co-cultured with 5 × 103 and 2.5 × 103 cells, respectively, in 96-well round-bottom plates in tissue culture media. TLR stimulation was performed with 1 µg/ml LPS (Escherichia coli; Sigma-Aldrich), 1 µM CpG 1668 (mouse), 1 µM CpG 2216 (human), or 1 µg/ml flagellin (Salmonella Typhi; InvivoGen). Cultures were incubated for 18 h. Supernatants were harvested for ELISA and remaining cells were incubated with Golgi Plug (BD) for 4 h and subsequently analyzed by flow cytometry.
Longman R.S., Diehl G.E., Victorio D.A., Huh J.R., Galan C., Miraldi E.R., Swaminath A., Bonneau R., Scherl E.J, & Littman D.R. (2014). CX3CR1+ mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22. The Journal of Experimental Medicine, 211(8), 1571-1583.
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3

Macrophage Cytokine and Nitric Oxide Assays

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Cells were seeded at 60,000 cells per well in 96-well CellBind plates (Corning) in macrophage media the day prior to stimulation. For cytokine analysis cells were stimulated with 1 µg/ml LPS (InvivoGen) or 1 uM CpG-1668 (InvivoGen, Tlrl-1668) for 16–20 hr. Cell-free supernatants were harvested and frozen at −20°C prior to analysis. For Griess assay analysis, cells were stimulated with 100 ng/ml LPS and 5 ng/ml IFNγ. After 24 hr cell-free supernatant was harvested and immediately analyzed.
Roberts A.W., Popov L.M., Mitchell G., Ching K.L., Licht D.J., Golovkine G., Barton G.M, & Cox J.S. (2019). Cas9+ conditionally-immortalized macrophages as a tool for bacterial pathogenesis and beyond. eLife, 8, e45957.
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4

Antibody Characterization and Immune Complex Analysis

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Antibodies used for flow cytometry are described in Extended Experimental Procedures. Chicken ovalbumin (OVA)-alexa555, bovine serum albumin (BSA)-alexa488, BSA-TRITC, DQ Green-BSA, 70kD TRITC-conjugated dextran and carboxylate-modified 2, 0.2 or 0.02 μm beads were purchased from Invitrogen (Carlsbad, CA). Endotoxin-free OVA (Hyglos) was conjugated to CpG 1668 or GpC 1668 oligonucleotides (both from Invivogen) as described in Extended Experimental Procedures. Polyclonal rabbit anti-OVA and polyclonal rabbit anti-BSA were purchased from AbD Serotec and Invitrogen respectively, and their affinity for murine FcγRs was determined with a Biacore X100 biosensor system as previously described (Kao et al., 2015 (link)), using a 1:1 Langmuir binding model as rabbits have only one IgG class (Rayner et al., 2013 (link)). Immune complexes containing OVA and BSA were prepared in vitro, as described in Extended Experimental Procedures, and analyzed by fast protein liquid chromatography using a ÄKTA FPLC system (GE Healthcare Life Sciences). Polyclonal rabbit anti-mouse serum albumin (#600-401-254), polyclonal rabbit anti-mouse transferrin (#600-401-255), polyclonal rabbit anti- mouse IgG (#610-4102) and polyclonal rabbit anti-mouse IgG F(ab′)2 (#310-4102) were purchased from Rockland Immunochemicals.
Stamatiades E.G., Tremblay M.E., Bohm M., Crozet L., Bisht K., Kao D., Coelho C., Fan X., Yewdell W.T., Davidson A., Heeger P.S., Diebold S., Nimmerjahn F, & Geissmann F. (2016). Immune Monitoring Of Trans-Endothelial Transport By Kidney Resident Macrophages. Cell, 166(4), 991-1003.
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