In the case of splenic macrophages, 10 units/mL IFN-γ was added to the culture medium to prime cells. In addition, 5 μg/mL polymyxin B was also added to avoid cell activation by LPS in the IFN-γ sample. Separately, RAW264.7 cells were incubated with pDNA/LA2000 complex for 2 h, then the cells were washed with RPMI-1640 CCI-779 solubility dmso and incubated with fresh growth medium for an additional 6 h, and the supernatants were collected for ELISA and kept at −80°C until use. PMDC05 cells were incubated with ODNs for 24 h, then the supernatants were collected for ELISA and kept at −80°C until

use. The level of murine TNF-α and murine IL-6 in the media was determined by ELISA using the OptEIA set (BD Biosciences Pharmingen, San Diego, CA, USA). The level of human TNF-α in the media was determined by human TNF-α ELISA set (eBioscience, San Diego, CA, USA). RAW264.7 cells were incubated with Alexa488-labeled ODN1668 with or without ODN1720 or DNase-treated ODN1720 for 4 h and MI-503 mouse washed

three times with PBS. Then, the intensity of cell fluorescence was analyzed by flow cytometry (FACScan; BD Biosciences) using CellQuest software (version 3.1; BD Biosciences). Cellular uptake was estimated by subtracting the mean fluorescence intensity (MFI) at 4°C from that at 37°C (ΔMFI) and was plotted against the incubation time. ODN1668 was mixed with DNase I- or DNase II-treated ODN1720. The mixture containing 0.5 μg ODN1668 was incubated with DNase I (2 units/μg ODN1668) or DNase II (3 units/μg ODN1668) at 37°C. After 0, 5, 15, 30 min of incubation (DNase I treatment) or 0, 20, 40, 80 min of incubation (DNase II treatment), the mixture Progesterone was placed on ice and the reaction was terminated by the addition of 3 μL 0.2 M EDTA solution per 10 μL of samples. The ODN samples were run on a 21% PAGE and stained with ethidium bromide. The image of the gel was recorded using LAS 3000 (Fujifilm Life Science, Tokyo, Japan) and analyzed using Multi Gauge software (Fujifilm Life Science). Differences in the cytokine release were statistically evaluated by Student’s t-test. Differences in the thickness

of mouse footpad were statistically evaluated by one-way analysis of variance (ANOVA) followed by the Tukey–Kramer test for multiple comparisons. A p-value of less than 0.05 was considered to be statistically significant. We wish to thank Dr. Hiroyuki Yoshitomi (Graduate School of Medicine, Kyoto University) for providing technical assistance for subcutaneous injection of ODN into the footpad of mice. This work is partly supported by the 21st Century COE Program “Knowledge Information Infrastructure for Genome Science” and by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Sciences and Technology, Japan. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”.