Fig. 3. Determination of TLR9-dependent activity of TLR9 agonists.

A In this reporter-based assay, TLR9-expressing HEK293-Blue cells were treated with ODN2395 and SD101 at increasing doses (0.004–10 µM) for 21 h. As a negative control, no-treatment (NT) and sequence control ODN5328 at 3 (C_3) and 10 (C_10) µM were used. The secreted embryonic alkaline phosphatase (SEAP) was determined by measuring the absorbance at 650 nm after addition of substrate. B Cells were pretreated with chloroquine (Chq, 1 µg/ml) for 45 min before the addition of ODN2395 or SD101 at increasing concentrations (0.012–3 µM) for 21 h, and absorbance was measured at 650 nm. All the experiments were performed at least three times with 2–3 replicates, and mean ± SEM was plotted in the graph.