CpG ODN (2006), TLR9 ligand (klasse B)

Sequence: 5'-tCGtCGttttgtCGttttgtCGtt-3' (Class B ODN 2006: 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.

Human

Functional Assay

1 mg/ml in endotoxin-free water

Upon receipt, store at -20°C (Stable for at least 6 months) . Avoid frequent freeze/thaw cycles.

A. CpG ODN 2006-mediated human TLR9 activation in TLR9/NF-kB LeeporterTM - HEK293 cells (Figure 2) . 1. Harvest TLR9/NF-kB LeeporterTM - 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 37°C in a CO2 incubator for overnight. 3. The next day, stimulate cells with various amounts of CpG ODN 2006. 4. Incubate at 37°C 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.