Que Inhibits T Cell Proliferation Figure 2. Que protectes the spinal cord from demyelination and loss of oligodendrocytes. A. MBP immunofluorescent staining displays an obvious decrease of myelin (arrows) in the white matter of the spinal cord in the untreated EAE controls (N = 5). Post-treatment with Que (N = 5) protects myelin from breakdown and displays a similar fluorescence intensity to the unimmunized controls (N = 5). B. Luxol Fast Blue (LFB) staining indicates a similar pattern after Que treatment (arrows). C. Que prevents the loss of CC1+ oligodendrocytes as compared with the untreated controls. Quantification of the observations is provided in the bar graphs. (*, p,0.05). Scale bar A = B = 0.5 mm, C = 0.2mm. doi:10.1371/journal.pone.0042746.g002 proper for Alex Fluor 488 (488 nm), Alex Fluor 568 (568 nm), Cy5 (628 nm) or DAPI (380 nm). For stereological quantification, serial sections of spinal cord from L1–L6 were collected (about 200 sections) and twenty sections were sampled from each animal in a systematic and random manner. In practice, every first and the tenth sections were sampled from the 200 sections systematically. After immunofluorescence staining, digital images of CD4+, CD8+, CC1+, NG2+, GFAP+, CD8+, CD11b+, CD68+ and MBP staining were acquired with a digital camera (Nikon, Japan) mounted on a 90 i fluorescence microscope (Nikon, Japan). The cell numbers of CD4, CD8, CC1 and NG2 positive cells and the mean intensity of CD11b+, GFAP+, CD68+, MBP+ and Fast Blue staining were quantified with Image-Pro Plus 5.0 (Media Cybernetics, Silver Spring, MD, USA). Flow Cytometry Analysis Mice were anesthetized, spleen and draining lymph nodes removed, and single cell suspension was prepared. Cells isolated from lymph nodes and spleens were adjusted at 16106 cells/vial and stained with Percp cy5.5-anti-CD4(clone RM4-5), PE-anti- CD25(clone PC61.5) or Percp cy5.5-anti-CD8 (clone 53-6.7,) for 30 min at 4uC, then washed with PBS containing 1% FCS. All antibodies were obtained from eBioscience. In some instances, isotype-matched IgG were used as negative controls. In each test, 1,000,000 cells were collected by a Canto II flow cytomerer using Cell Quest Diva software (BD Biosciences) and analyzed by FlowJo software (TriStar). CFSE Proliferation Assay Proliferation of T cells was measured by dilution of the dye CFSE. To detect MOG35–55-specific proliferation of T cells, single cell suspensions from spleen were obtained at 10 days after MOG35–55- immunization. Cells were incubated with CFSE (2 mM) (eBioscience) for 10 min at 37uC in the dark, then washed with cold complete media 3 times. Cells labeled with CFSE were cultured in 96-well plates (26105 cells/well) and stimulated with indicated concentration of MOG35–55 peptide in triplicate at 0, 1, 10 or 100 mg/ml. In additional experiments, CD4+CD252 T cells in spleens of na¨ıve C57BL/6 mice (N = 10) were fractionated using magnetic bead chromatography (Miltenyi). Purity of the samples was routinely tested after sorting and was .95%. Cells were the labeled with CFSE and cultured in 96-well plates (26105 cells/well) coated with anti-CD3 antibody (1 mg/ml) by adding Que (1 mM) or not for 72 h. The results were analyzed using FACS (Canto II, BD). RNA Preparation and Real-time PCR CD4+CD252 T cells were cultured in 96-well plate coated with anti-CD3(1 mg/ml), incubating with/wihout Que(1 mM) for differ- ent time (0, 12, 24 or 48 h), total RNA was extracted with Trizol Reagent (Invitrogen) following the manufacture’s instructions after indicated time. Reverse transcription of RNA was performed using PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e42746