FIG. 2.

CpG 2395, DNA 2395, and TNA 2395 activate Ramos and Raji cells, while only CpG 2395 induces SEAP reporter activity in HEK-Blue hTLR9 cells. (A, B) Ramos and Raji cells were plated at a density of 2 × 10⁵ cells in 12-well plates and stimulated with the indicated ligands at the indicated concentrations. After 72 h, cells were harvested for flow cytometric analysis of surface-expressed CD86 expressing using a PE-labeled antibody specific for CD86. A PE-labeled isotype antibody was included as a control. Data are shown relative to the untreated control for Ramos (A) and Raji (B) cells. Error bars represent the standard deviation between two replicates within a single experiment. A representative experiment is shown in each panel. Experiments were performed 3 times. (C, D) HEK-Blue hTLR9 cells were plated at a density of 5 × 10⁴ in 96-well plates and allowed to adhere overnight. Cells were then stimulated with the indicated ligands at the indicated concentrations. After 24 (A) or 72 (B) hours, supernatants were harvested and tested for SEAP activity using QUANTI-Blue reagent. Samples were assayed in triplicate. Error bars represent standard deviations for three replicates within a single experiment. A representative experiment is shown in each panel. Experiments were performed 3 times. PE, phycoerythrin; SEAP, secreted alkaline phosphatase.