Samples were analyzed using a BD FACS Calibur flow cytometer and data were analyzed using FlowJo software. Isotype-matched RG7422 PE- and FITC-conjugated mAbs of irrelevant specificity were used
as negative controls. Lymphocytes from either EAMG or CFA control rats (2 × 106/mL) were cultured in the presence of AChR R97-116 (10 μg/mL) with or without CGS21680 (30 nM). After a 72 h incubation, supernatants were collected and IFN-γ, IL-4, IL-17, and TGF-β levels were determined using respective ELISA kits (Shanghai Senxiong Biotech Industry Co. Ltd., China) according to the manufacturer’s instructions. The analyses were performed in triplicate and the results are expressed as the mean cytokine concentration (pg/mL) ± SD. For preventive treatment experiments, rats were given CGS21680 (0.5mg/kg intraperitoneally (i.p.)) in PBS starting 1 day before EAMG induction and every 3
days throughout the course of the experiment. Therapeutic treatment of EAMG consisted of 1.0 mg/kg CGS21680 administered Small molecule library clinical trial i.p. every 3 days starting 29 days post immunization. Hind limb muscles were harvested, snap frozen in liquid nitrogen, and a cryostat used to generate 10-μm thick sections. We incubated the sections with biotin-conjugated goat antirat IgG (Sigma-Aldrich) for 1 h. Sections were then stained with tetramethylrhodamine-labeled α-BTX (Molecular Probes), FITC-labeled goat antirat C3 (Nordic Immunological Laboratories), and Alexa Fluor 350-labeled streptavidin. Sections were then analyzed using a fluorescence microscope (LSM 700, Zeiss). Data were expressed as mean ± SD. Differences between groups were analyzed using Graphpad software (Graphpad software, CA) and the two-tailed Student’s t-test for paired and unpaired data. p < 0.05 were considered Arachidonate 15-lipoxygenase statistically significant. This work was supported by Heilongjiang Provincial Innovation Found for Postgraduates (YJSCX2011-324HLJ, Na Li is the recipient), National Nature Science Foundation of China (81000511, Lili Mu is the recipient), China Postdoctoral
Science Foundation (20100480062, Lili Mu is the recipient), National Nature Science Foundation of China (81000536, Qingfei Kong is the recipient), China Postdoctoral Science Foundation (20100471094, Qingfei Kong is the recipient), National Nature Science Foundation of China (30901330, Bo Sun is the recipient), National Nature Science Foundation of China (81100883, Yumei Liu is the recipient), and the Harbin Medical University Cell Biological Engineering Center (1151gzx05). The authors declare no financial or commercial conflict of interest. Disclaimer: Supplementary materials have been peer-reviewed but not copyedited. Figure S1. The A2ARagonist CGS21680 reduced the number of AChR antibody-secreting cells. Figure S2.