Mhcii pe

The above isolated cells were blocked with 10 % normal rat serum/PBS on ice for 30 min. The brain mononuclear cells were stained with a combination of anti-mouse antibodies CD206-FITC + MHCII-PE + F4/80-PE/Cy7 + CD45-APC and the splenocytes with a combination of CD11b-FITC + CD45-PE + F4/80-PE/Cy7 + Gr-1-APC or CD206-FITC + MHCII-PE + F4/80-PE/Cy7 + CD45-APC (all from BioLegend) on ice, protected from light, for 30 min. Cells were washed and resuspended in 500 μl PBS/1 % FCS/0.02 % NaN₃. The flow cytometric data were acquired with a two-laser, six-color Gallios flow cytometer and analyzed by Kaluza analysis 1.3 software (Beckman Coulter). Brain mononuclear cells were defined as follows: microglia CD45⁺F4/80⁺, macrophages CD45^hiF4/80⁺, M1-type macrophages CD45^hiF4/80⁺MHCII⁺CD206⁻, and M2-type macrophages CD45^hiF4/80⁺MHCII^−/+CD206⁺. Splenocytes were defined as follows: granulocytes CD45⁺F4/80^−/+Gr-1⁺⁺, monocytes CD45⁺F4/80⁻Gr-1⁺, monocyte-derived macrophages CD45⁺CD11b⁺F4/80⁺Gr-1⁻, tissue-resident macrophages CD45⁺F4/80⁺⁺Gr-1^−/+, M1-type macrophages CD45⁺F4/80⁺MHCII⁺CD206⁻, and M2-type macrophages CD45⁺F4/80⁺MHCII^−/+CD206⁺. Cell populations were calculated as percentages among total leukocytes or macrophages.

The following antibodies were used to characterize BMDCs and assess their activation state: MHC II-PE, CD11c-APC, CD86-PE-Cy7, CD80-FITC and CD11b-APC-Cy7 (all from BioLegend). Acquisition was performed on an Attune NxT (ThermoFisher). Leukocyte populations from BM, liver and spleen were also analyzed by flow cytometry. Single cells were excluded from dead cells using the LIVE/ DEAD Zombie NIR Fixable Viability Kit (BioLegend). Immuno-phenotyping was performed using the following antibodies: CD45-BV510, CD49b-PE-Dazzle, CD19-PerCP, CD3-FITC, CD4-AF700, CD8-BV785, C44-BV650 and CD62L-BV421 (all from BioLegend). Full minus one (FMO) controls were used to determine positivity. Precision count beads (BioLegend) were used to count immune cells in different organs. Before acquisition, stained cells were fixed with 1% Paraformaldehyde (Sigma-Aldrich). Acquisition was performed using the BD LSRFortessa and data were analyzed using the FlowJo software (TreeStar) version 10.

Cells were characterized by flow cytometry using the monoclonal antibodies (mAbs) CD11b/c-APC, CD80-PE, CD86-PE, MHCI-FITC, and MHCII-PE (BioLegend, San Diego, CA, USA). For analysis of the glycocalyx, lectins from Maackia amurensis (MAL II, indicating α2-3 Sia) and Sambucus nigra (SNA-I, indicating α2-6 Sia) were used (Vector Labs). Lectins were biotin conjugated. PE-streptavidin was used for detection. Negative controls for non-specific fluorescence were used, these consisted of PE-streptavidin staining solutions in the absence of the lectin conjugated to biotin. Lectins were prepared in lectin staining buffer (PBS containing 1% FBS, 1 mmol/L CaCl₂, and 2 mmol/L MgCl₂) and resuspended in FACS buffer (PBS containing 2% fetal calf serum and 0.01% NaN₃, all from Sigma-Aldrich) before analysis using a FACS Canto II (BD Biosciences, Oxford, UK).
For analysis of the assays involving lymphocytes from the lymph nodes and spleen, the following mAbs were used CD3/PE, CD8/PE-Cy7, CD4/APC (BioLegend), and CD25/FITC (eBioscience, San Diego, CA, USA). Prior to staining, cells were washed with FACS buffer. mAbs were diluted in 50 µL FACS buffer, added to the cells, and incubated for 15 min at 4°C. To remove any unbound antibodies, the cells were washed three times with FACS buffer. The cells were then filtered through a nylon mesh (40 µm) before analysis in the cytometer.