CpG ODN (C274), TLR9 ligand (Class C)
Fig-1: Induction of human TLR9 activity by CpG ODN C274.
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| Amount : | 0.1 mg |
| Content : | 1 mg/ml in entotoxin-free water |
| Storage condition : | Upon receipt, store at -20°C (Stable for at least 6 months). Avoid frequent freeze/thaw cycles. |
Sequence: 5'-tCGtCGaaCGttCGagatgat-3' (Class C ODN C274: Bases are phosphorothioate.)
Synthetic oligodeoxynucleotides (ODN) containing unmethylated deoxycytosine-deoxyguanosine (CpG) motif are equivalent to bacterial DNA in the immunostimulatory activity, which can induce innate immunity via Toll-like receptor 9 (TLR9) in mammals. There are three major classes of CpG ODNs, and each ODN class exhibits different stimulatory effects on immune cell activation. Class A ODNs are potent inducers of IFN-alpha that leads to the plasmacytoid dendritc cell (pDC) maturation. Class B ODNs are relatively weak inducers of type I IFNs but strong stimulators of human B cells and monocyte maturation. Class C ODNs combine elements of both Classes A and B ODNs, which can induce IFN-alpha in pDC and activation of B cells.
A. CpG ODN C274-mediated human TLR9 activation in TLR9/NF-kB Leeporter™ – HEK293 cells (Figure 2).
1. Harvest TLR9/NF-kB Leeporter™ – HEK293 cells and seed cells into a white solid-bottom 96-well microplate in 100 ul of growth medium at 5 x 104 cells/well.
2. Incubate cells at 37oC in a CO2 incubator for overnight.
3. The next day, stimulate cells with various amounts of CpG ODN C274.
4. Incubate at 37oC in a CO2 incubator for 6-16 hours.
5. Add 30-50 ul of luciferase assay reagent per well.
6. Incubate at room temperature for 1-5 minutes and measure luminescence using a microplate luminometer.
B. CpG ODN C274-mediated mouse TLR9 activation in NF-kB Leeporter™ – RAW 264.7 cells (Figure 3).
1. Harvest NF-kB Leeporter™ – RAW 264.7 cells and seed cells into a white solid-bottom 96-well microplate in 100 ul of growth medium at 5 x 104 cells/well.
2. Incubate cells at 37oC in a CO2 incubator for overnight.
3. The next day, stimulate cells with various amounts of CpG ODN C274.
4. Incubate at 37oC in a CO2 incubator for 6-16 hours.
5. Add 30-50 ul of luciferase assay reagent per well.
6. Incubate at room temperature for 1-5 minutes and measure luminescence using a microplate luminometer.
For Research Use Only. Not for use in diagnostic/therapeutics procedures.
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