cc-3′; and for IL-1, forward 5′-agttgacggaccccaaaag-3′ and reverse 5′-agctg gatgctctcatcagg-3′. Cycle threshold values were normalised to GAPDH gene expression using GAPDH primers, forward 5′-gggttcctataaatacggactgc-3′ and reverse 5′- ccattttgtctacgggacga-3′. The PCR system was 95 for 5 min followed by 40 cycles of 95 for 10 s, 60 for ten s and 72 for 10 s. Final 
results are expressed as relative fold adjust as calculated by the delta delta CT strategy [24].
To confirm that S100B increases CCL22 production by RAW 264.7 macrophages or BMDM, supernatant was collected immediately after 24 h incubation with or without S100B, centrifuged at 167 g for 5 min at four and stored at -80. Supernatants had been analysed for CCL22 working with a DuoSet ELISA kit (R&D Systems) as recommended by the manufacturer. For studies on IL-1 pro-form production, RAW 264.7 cells or BMDM were treated with 2 M S100B and five g/ml Brefeldin A for 4 h (Sigma Aldrich, UK). Cells have been harvested in 2% FCS 19569717 in PBS for flow cytometric analysis of intracellular pro-IL-1. Cells were incubated on ice for 20 min with Fc block (4 g of CD16/CD36, BD Biosciences, Oxford, UK) before surface staining with PerCP-CY5.five rat monoclonal anti-mouse CD11b (BD BioSciences, 0.five l of 200 g/ml). Cells have been then fixed and permeabilised in BD BioSciences fixative and permeabilisation buffer, following manufacturer guidelines. PE-conjugated rat anti-mouse IL-1 pro-form (eBioscience Hatfield, UK at 0.06 g per test in permeabilsation buffer) was added and incubated at room temperature for 30 min in the dark. Cell suspensions had been then washed twice in permeabilisation buffer, mixing before each wash, with a final wash in FACS buffer (2% v/v FCS in PBS containing 2% w/v sodium azide). Cells were re-suspended in 300 l FACS buffer before data acquisition on the LSR II flow cytometer (BD BioSciences). Analysis of FACS data was carried out utilizing FlowJo Software, (Treestar, OR, USA).
EAU was induced utilizing a standard approach [25]. Both S100B KO and C57BL/6 mice were immunised subcutaneously in 2 thighs (50 l/thigh) with a total concentration of 500 g per mouse retinol binding protein-3 peptide ten (RBP-31-20; GPTHLFQPSLVLDMAKVLLD; New England Peptide LLC, MA, USA) emulsified with Complete Freunds Adjuvant (CFAH37Ra, BD Biosciences,) containing additional 2.5 mg/ml Mycobacterium tuberculosis (1:1 v/v, BD Biosciences). An additional 100 l, ten g/ml Bordetella pertussis toxin was administered by intraperitoneal injection. Mice were sacrificed by CO2 asphyxiation and cardiac puncture and eyes collected.
Disease progression was followed applying Topical Endoscope Fundal Imaging (TEFI) at day 15, day 21 and day 24 post peptide immunization (pi) to compare S100B KO and C57BL/6 WT mice. Mice were Ro 41-1049 (hydrochloride) cost anaesthetised, pupils dilated utilizing 1% (w/v) tropicamide and 2.5% (w/v) phenylephrine hydrochloride (Bausch & Lomb Minims, Chauvin Pharmaceuticals Ltd, London, UK), and Viscotear liquid gel (Novartis Pharmaceuticals, Frimley, UK) was applied to each eye to provide good endoscope contact to cornea and avoid eyes drying. Fundus images obtained had been clinically graded on a scale 0 by scoring changes in retinal infiltrate and lesions, retinal vessels and clarity of the optic disc according to Xu et al.,2008 [26]. At approximately peak disease, day 24 pi, mice were culled and eyes had been snap frozen in OCT compound (Tissue-Tek, Agar Scientific, Essex, UK). Eyes were serially sectioned at 6 m and sections fixed in acetone, haematoxylin stained and dehydrated in ethanol. Se