CpG ODN (C274), TLR9 ligand (Class C)

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 37^oC in a CO₂ incubator for overnight.

 

3. The next day, stimulate cells with various amounts of CpG ODN C274. 

 

4. Incubate at 37^oC 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.