For CXCR5 staining, cells were treated with unconjugated anti-CXCR5 antibody at 37?C for 1?h followed by secondary antibody at RT for 30?min

For CXCR5 staining, cells were treated with unconjugated anti-CXCR5 antibody at 37?C for 1?h followed by secondary antibody at RT for 30?min. T cells in autoimmune demyelination. H37 Ra CD200 (BD). On day 0 and 2 post immunization, mice were intravenously (i.v.) injected with 0.2?g Pertussis toxin (List Biological Laboratories) in 0.2?ml PBS. Treated mice were monitored daily and the disease score was determined as follows: 0: no clinical sign, 1: weakness of the tail, 2: complete tail paralysis, 3: partial hind limb paralysis, 4: complete hind limb paralysis, 5: incontinence and partial or complete paralysis of forelimbs, 6: death [35]. 2.3. Histology Animals were anesthetized and transcardially perfused with PBS followed by zinc formalin. Brains, spinal cords, and LNs (axillary, brachial and inguinal) were removed, fixed in zinc formalin, and paraffin embedded. Sections were stained with hematoxylin and eosin. Alternatively, fixed spinal cord sections were deparaffinized and hydrated with 95% EtOH, followed by staining with Luxol Fast Blue solution at 56CC58?C overnight. Stained sections were then washed with 95% EtOH and H2O before differentiation with lithium carbonate and 70% EtOH. Images were acquired using a BX61 light microscope (Olympus) Mutant IDH1-IN-1 and CellSens software (Olympus). The percentage of demyelination (% demyelinated/total white matter of the spinal cord) was determined using ImageJ 64 (NIH) software. 2.4. Confocal microscopy Tissues were harvested as described above and fixed with 4% PFA at 4?C for 4?h, followed by immersion in 10%, 20%, 30% sucrose-PBS for 12?h each. 5C15?m thick sections were then prepared from OTC-embedded samples and fixed in acetone for 10?min?at 4?C. For staining, sections were blocked with goat serum for 2?h at room temperature (RT) and treated with primary antibodies (rat anti-mouse CD3 (CD3-12), hamster anti-mouse CD11c (N418), rabbit anti-mouse cleaved caspase 3 (Abcam)) prior to incubation overnight at 4?C. After washing Mutant IDH1-IN-1 with PBS, samples were exposed to secondary Mutant IDH1-IN-1 antibodies (Alexa 647-goat anti-rabbit IgG, Alexa 488-goat anti-hamster IgG, Alexa 568-goat anti-rat IgG, Abcam) for 30?min. Finally, slides were overlaid with DAPI (Vector) and examined with a confocal microscope (Zeiss 710). 2.5. Antibodies and flow cytometry The following monoclonal antibodies were used: PE or PerCP-Cy5.5-conjugated rat antiCmouse CD4 (RM4-5), FITC-conjugated rat antiCmouse CD8 (53C6.7), FITC or e450-conjugated hamster antiCmouse CD11c (HL3), PerCP-Cy5.5-conjugated mouse antiCmouse Ly-6C (HK1.4), APC-conjugated rat antiCmouse F4/80 (BM8), FITC-conjugated rat antiCmouse IL-1 (NJTEN3), PerCP-Cy5.5-conjugated mouse anti-mouse Foxp3 (FJK-16s) and rat antiCmouse CD16/32 (2.4G2) (eBioScience); PerCP-Cy5.5-conjugated mouse antiChuman CD4 (RPA-T4), PerCP-Cy5.5-conjugated hamster antiCmouse CD3 (145-2C11), Brilliant Violet 421-conjugated mouse anti-mouse CD45.1 (A20), APC-conjugated mouse anti-mouse CD45.2 (104), PE-Cy7-conjugated mouse anti-mouse NK1.1 (PK-136), PerCP-Cy5.5 or PE-conjugated rat antiCmouse CD45 (30-F11), APC-Cy7-conjugated mouse anti-mouse MHC-I (28-8-6), Brilliant Violet 510-conjugated mouse anti-mouse MHC-II (M5/114.15.2), PE-conjugated mouse anti-mouse CD80 (2D10), PE-Cy7-conjugated mouse anti-mouse CD83 (HB15e), PE-Cy7-conjugated mouse anti-mouse PD-1 (RPM1-30), PE-conjugated mouse anti-mouse Fas (SA367H8), APC/Fire 750-conjugated goat anti-rat IgG (poly4054), Alexa Fluor 488-conjugated rat anti-mouse IL-2 (JES6-5H4), APC-conjugated rat antiCmouse IL-6 (MP5-20F3), Alexa Fluor 647-conjugated rat antiCmouse IL-10 (JES5-16E3), PE-conjugated rat antiCmouse IL-12 (C15.6), Alexa Fluor 647 or APC-conjugated rat antiCmouse IFN- (XMG1.2), PE-conjugated mouse anti-mouse IL-17F (9D3.1C8), APC-conjugated rat anti-mouse IL-17A (TC11-18H10.1) and APC-conjugated rat anti-mouse TNF (MP6-XT22) (BioLegend); rat anti-mouse CXCR5 (2G8), FITC-conjugated mouse anti-mouse B220 (RA3-6B2), PE-conjugated rat anti-mouse Ly-6G (1A8), FITC-conjugated rabbit anti-mouse caspase-3 (C92-605), FITC-conjugated rat anti-mouse CD86 (GL1) (BD); rabbit anti-mouse cleaved caspase 3 (Abcam); PE-conjugated rat anti-mouse CCR2 (475,301) (R&D). For surface staining, 106?cells were blocked with 1?g of anti-CD16/32 antibody and stained with the indicated antibodies at 4?C. For CXCR5 staining, cells were treated with unconjugated anti-CXCR5 antibody at 37?C for 1?h followed by secondary antibody at RT for 30?min. For intracellular staining, cells were fixed using Cytofix Solution (BD) and stained for Foxp3 or intracellular cytokines. To detect antigen-specific T cells, 106?cells were cultured in a 96-well round bottom plate in the presence of Brefeldin A (BFA, Invitrogen) and MOG35-55 peptide (Bio-Synthesis) for 6C12?h. To determine the absolute number of cells, CountBright? absolute counting beads (Invitrogen) were added during staining. A Dead Cell Apoptosis Kit with Annexin V FITC and PI (Thermo Fisher) was used to gate live cells. Flow cytometric data were acquired using a FACSVerse (BD) and were analyzed using FlowJo software (Tree Star). 2.6. CD11c+ cell separation using Mutant IDH1-IN-1 magnetic beads and adoptive transfer Spleen single-cell suspensions.

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