Anti 1 a 1 e bv605

Single cell suspensions from IEL and LP were labelled with a cocktail of flurochrome conjugated antibodies against surface antigens purchased from either BD Biosciences (San Jose, CA) or Biolegend (San Diego, CA): anti-CD45 FITC, anti-CD19 PerCP Cy5.5, anti-Siglec H Pacific Blue, anti-I/A I/E BV605, anti-CD11b BV 650, anti-CD3e BV786, anti-B220 BUV496, anti-EpCam APC, anti-F4/80 AF700, anti-Ly6c APC-Cy7, anti-CD11c PE-CF594, anti-CD103 PE-Cy7 and Zombie aqua kit. Cells were fixed with 1% PFA prior to acquisition in Flow Cytometry. Cells were acquired using a 18 color BD LSRFortessa (San Jose, CA) and analyzed with FlowJO software (Ashland, Oregon).

For flow cytometry analysis, isolated brain cells were pre-incubated for 15 min with purified anti-mouse CD16/32 (BioLegend, San Diego, CA, USA) to block Fc-mediated non-specific binding of antibodies. Then, cells were stained with the following antibodies: anti-CD45-BV421, anti-CD3ε-PE/Dazzle 594, anti-CD19-PE, anti-CD4-PerCP/Cy5.5, anti-CD8a-PE/Cy7, anti-CD69-APC/Cy7, anti-H-2-FITC, anti-CD317-PE, anti-Ly6C-APC-Cy7, anti-I-A/I-E-BV605 (all from BioLegend), anti-CD11b-AF700 (BD Biosciences, Franklin Lakes, NJ, USA), anti-CD45R/B220-APC (Thermo Fisher Scientific), and anti-CD11c-PE/Cy7 (Tonbo Biosciences, San Diego, CA, USA) for 20 min on ice. After surface staining, dead cells were stained with Zombie Aqua™ Fixable Viability Kit (BioLegend).
Data were acquired on a FACS LSR Fortessa (BD Biosciences), and the percentage of each cell population and mean fluorescence intensity were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA). For the analysis of cell numbers of a specific population, total brain cells were visually counted after density gradient centrifugation as described above; then, actual numbers were calculated using the percentages from flow cytometric analysis.

For flow cytometry analysis, isolated cells were pre-incubated for 10 minutes with purified anti-mouse CD16/32 (BioLegend) to block the Fc-mediated non-specific binding of antibodies. Then, cells were stained with the following antibodies: anti-CD64-PE/Dazzle 594, anti-CD115-PE/Dazzle 594, anti-CD3-PerCP/Cy5.5, anti-NK1.1-PerCP/Cy5.5, anti-Ly6G-PerCP/Cy5.5, anti-CD24-PE/Cy7, anti-CX3CR1-APC, anti-CCR5-APC, anti-Ly6C-APC/Cy7, anti-CD45-BV421, anti-I-A/I-E-BV605 (all from BioLegend), anti-CD19-PerCP/Cy5.5, anti-CD11b-AF700 (BD Biosciences, Franklin Lakes, NJ, USA), anti-CCR2-APC (R&D Systems, Minneapolis, MN, USA), and anti-F4/80-PE (Thermo Fisher Scientific) for 20 minutes on ice. After surface staining, dead cells were stained with Zombie aqua™ Fixable Viability Kit (BioLegend). For the intracellular staining of TLR7, cells were fixed and permeabilized with the BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) according to the manufacturer’s instructions. Then, cells were stained with anti-TLR7-PE (BD Biosciences). Data were acquired on a FACS LSR Fortessa (BD Biosciences) and the percentage of each cell population and mean fluorescence intensity were analyzed using FlowJo software (TreeStar Inc, Ashland, OR, USA).