Splenocytes were cultured in anti-CD3 coated flat-bottom 96-well plates (0.5 × 106 cells/well) in the presence of increasing concentrations (0–1000 ng/mL) of the immunosuppressive drug MP . For MOG35-55 stimulation, splenocytes were harvested from EAE mice, cultured at 0.5 × 106 cells/well in a U-shape 96-well plates and stimulated with 10 μg/mL MOG35-55. Culture plates were incubated at 37°C in a 5% CO2 atmosphere. After 48 h incubation, supernatants were harvested and stored at −80°C until cytokine analysis. Levels
of IL-2, IFN-γ, IL-4, IL-6, IL-10, IL-1, TNF-α, MCP1, and IL-17A were measured either with a multiplex ELISA kit (Quansys Biosciences, Logan, Utah) or with individual cytokine sandwich ELISA kits (Biolegend, San Diego, CA) as indicated in figure legends and according to manufacturer’s instructions. The immunosuppressive effect of MP is presented as percent of cytokine production without NVP-AUY922 nmr MP. Mice were immunized by subcutaneous injection
into flanks of 100 μg MOG35-55 emulsified in CFA (Difco, Detroit, MI). Pertussis toxin (List Biological Laboratories, Campbell, CA) was injected intraperitoneally (500 ng/mouse) selleck immediately following MOG35-55 injection and again 48 hours later. From day 9 postimmunization, mice were examined daily for clinical signs of the disease and the manifestation of the disease was graded on a 0–5 scale according to the following parameters: 0 = no clinical signs; 0.5 = loss of tail tonus; 1 = tail paralysis; 2 = partial hind-limb paralysis; 3 = hind-limb paralysis; 4 = complete paralysis; 5 = death. All statistical analyses were performed with
GraphPad Prism version 5.02 for Windows (GraphPad Software, San Diego, CA). All variables are expressed as mean ± SEM. p-values were calculated with Student’s t-test or ANOVA test as indicated in figure legends. We thank Dr. Tali Brunner and Prof. Marta Weinstock-Rosin for their valuable comments. We thank Dr. Irit Solodkin for graphical editing the manuscript figures. The Israel Science Foundation and Israel Ministry of Health supported this study. The authors declare no financial or commercial conflict of interest. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized TCL for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Fig. 1. CVS induces anxiety-like behaviors. Anxiety levels were quantified following 24 days of CVS or nonstress conditions. The elevated plus maze (A–B) and open field tests (C) were performed as described in Materials and methods. Bar graphs represent means ± SEM of 20–21 mice in each group, pooled from three independent experiments. p-values were calculated by Student’s t-test. **p < 0.01; ***p < 0.001.