CpG ODN (1585), TLR9 ligand (Class A)

A. CpG ODN 1585-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 μl 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 1585. 4. Incubate at 37°C in a CO2 incubator for 6-16 hours. 5. Add 30-50 μl 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 1585-mediated mouse TLR9 activation in NF-kB LeeporterTM – RAW 264.7 cells (Figure 3) . 1. Harvest NF-kB LeeporterTM – RAW 264.7 cells and seed cells into a white solid-bottom 96-well microplate in 100 μl 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 1585. 4. Incubate at 37°C in a CO2 incubator for 6-16 hours. 5. Add 30-50 μl of luciferase assay reagent per well. 6. Incubate at room temperature for 1-5 minutes and measure luminescence using a microplate luminometer.