Supplementary MaterialsAdditional file 1: Figure S1. C. Sorted cells are too rare to allow reliable re-analysis (most re-analyzed cells are found in the gate containing dead cells and debris). (PDF 354?kb) 12974_2017_915_MOESM2_ESM.pdf (354K) GUID:?2BF7D4BD-0550-4EEF-AC9C-ACB4D3F19E92 Additional file 3: Figure S3. MHC class II retinal expression is highly induced during classical EAU and adoptive transfer EAU, both during induction and at disease peak. Eye cryosections were stained for MHC class II (green) and IBA1 (red) detection 21?days after classical EAU induction (B), 14?days (C) or 21?days after adoptive transfer (AT) (D). Naive eyes were used as control (A). In each picture, quantification was made with the co-staining module of the Imaris 7.3 software. Each cell was counted individually. Results are expressed as the percentage of IBA1+ or MHCII+ single positive cells and IBA1+MHCII+ double-positive cells among the total of single and double-positive cells. Clevudine The DIC image was added to better localize the RPE. A. MHC class II expression in na?ve eyes. B. MHC class II expression during classical EAU at day 21. Clevudine C. MHC class II expression during AT EAU at day 14. D. MHC class II expression during AT EAU at day 21. (PPTX 3600?kb) 12974_2017_915_MOESM3_ESM.pptx (3.5M) GUID:?07CD9986-4A93-4753-BABB-5FFDF77A7B85 Additional file 4: Figure S4. MHC class II expression in the retina during classical EAU. Three weeks after immunization, eye cryosections were prepared and stained for MHC class II (green) and IBA1 (red) or endoglin (magenta) detection. Cell nuclei were stained with Hoechst (blue). Each picture Clevudine was chosen as representative of an experiment conducted on six or more animals. A. MHC class II and IBA1 expression. B. MHC class II and endoglin expression. (PPTX 7276?kb) 12974_2017_915_MOESM4_ESM.pptx (7.1M) GUID:?8D8038DB-4024-4C48-909A-9D33EBD016CE Additional file 5: Figure S5. Kinetics of co-stimulatory molecule expression by MHC class II cells during classical EAU and adoptive transfer EAU. Fourteen or 21?days after disease induction, the retinas were carefully dissected, cut into small pieces, and dissociated by enzymatic digestion. The single-cell suspensions, excluding dead cells (DAPI+), were analyzed by flow cytometry for MHC class II, CD80, CD86, and CD40 expression using fluorochrome-conjugated-specific antibodies. Data are representative of three independent animals for each disease model and timepoint, matched for disease grade. Only MHC class II+ cells are shown. A. Classical EAU, day 14. B. Classical EAU, day 21. C. Adoptive transfer EAU, day 14. (PPTX 2433?kb) 12974_2017_915_MOESM5_ESM.pptx (2.3M) GUID:?639CC951-1E6E-4801-95D0-0D521F975CFF Additional file 6: Figure S6. Kinetics of MHC class II and hematopoietic cell marker expression on the three types of potential APCs during classical EAU and adoptive transfer EAU. Fourteen or 21?days after disease induction, retinas were carefully dissected, cut into small pieces, and dissociated by enzymatic digestion. The single-cell suspensions, excluding inactive cells (DAPI+), had been analyzed by stream cytometry for MHC course II, Compact disc45, Compact disc11b, and Ly6C appearance using fluorochrome-conjugated particular antibodies. Data are representative of three unbiased animals for every disease model and timepoint, matched up for disease quality. Data symbolized: Mean??SEM. For every histogram, groups had been likened using Kruskal-Wallis lab tests (all beliefs 0.05). A. Percentage of MHC course II+ cells in the retina during traditional EAU or adoptive transfer (AT) EAU, at time 14 or time 21. B. Percentage of hematopoietic Compact disc45+Compact disc11b+ cells among MHC course II+ cells in the retina during traditional EAU or AT EAU, at time 14 or time 21. C. MFI for MHC course II appearance by hematopoietic or non-hematopoietic cells in the retina during traditional EAU or AT EAU, at time 14 or time 21. D. Percentage of Ly6C+ cells among hematopoietic MHC course II+ cells in the retina during traditional EAU or AT EAU, at time 14 or time 21. (PPTX 57?kb) 12974_2017_915_MOESM6_ESM.pptx (58K) GUID:?FDE23C22-B9B2-4888-BEDA-4EE0DF39FFB0 Extra document 7: Figure S7. Evaluation of MHC course II appearance in retinal wholemounts during adoptive transfer EAU. Three weeks after adoptive transfer, the eye were gathered and the complete retinas had been dissected and stained for MHC course II (green) and endoglin (magenta) recognition. Retinas from three unbiased animals had been stained in a single test. A. MHC course II KIR2DL5B antibody and endoglin appearance on the ora serrata. B. MHC course II and endoglin appearance in the central retina. C. MHC course II and endoglin appearance around the.