Cd8a apc

Single-cell suspensions were blocked with mouse FcR blocking reagent (Miltenyi Biotec) for 10 min at 4 °C prior to surface staining. Cell viability was assessed by Fixable viability dye eFluor 520 (eBioscience) to exclude dead cells. The following anti-mouse antibodies were used: FITC-CD11b, PE-F4/80, APC-F4/80, FITC-CD45, PerCP-eFluor 710-MHC Class II (I-A/I-E), PerCP-eFluor 710-CD3, FITC-CD3e, FITC-CD4, APC-CD4, APC-CD8a, PE-PD-L2, PE-B7-H2, PE-B7-H3, PE-B7-H4, PE-TIM-4, PE-VISTA from eBioscience; APC-CD206, PE-PD-L1, APC-CD86, PE/Cy7 Ki-67 from Biolegend; V450-CD4, BV510-CD8, PE-IFN-γ from BD. The following anti-human antibodies were used: PerCP-eFluor 710-CD3, APC-CD4, APC-CD8a from eBioscience; FITC-CD14, PE-PD-L1, APC-CD163, APC-CD86, FITC-HLA-DR, PE-Cy7-CD163, PE-CD25, PE-IFN-γ from Biolegend; FITC-CD8, PE-IL-10 from BD. For intracellular staining, cells were fixed and permeabilized with the Fixation/Permeabilization solution kit (BD). All flow cytometry data was acquired on FACSCalibur or LSRFortessa (BD, San Jose, USA) and analyzed by FlowJo V10 (TreeStar, Ashland, USA).

Tissues of 5 CD mice and 10/11 HFrD mice of each cohort were analyzed by flow cytometry for immune cells, using two panels for myeloid and T cell detection. Tissues were processed for flow cytometry analysis, as previously described [29 (link)] and stained with the following antibodies: APC-anti-F4/80, APC-e780-anti-cd11b1β, Alexa700-anti-Ly6G, eFluor450-anti- CD45, PE-Ly6C, eFluor450-anti-CD4, APC-CD8a, FITC- anti-CD3, APCe780-NK1.1, and PE-anti-gamma delta TCR (all eBioscience, San Diego, CA, USA), with the addition of 7-AAD to quantify live cells. Flow cytometry data were acquired on a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA) and analyzed using FlowJo™ Software (Becton, Dickinson, and Company, Franklin Lakes, NJ, USA) using compensation and gating templates previously generated (Supplementary Figure S5). Cell populations are represented as the percentage of cells compared with all CD45+ cells.

Mononuclear cells were isolated from brain and spinal cord of CX3CR1-WT, CX3CR1-KO, and hCX3CR1^I249/M280 mice by homogenization of CNS tissues as previously described (Pino and Cardona, 2011 (link)). Cellular suspensions were blocked using anti-mouse CD16/CD32 (clone 2.4G2, BD Pharmingen) followed by incubation for 30 min with a mix of fluorochrome-conjugated anti-mouse antibodies as follows. Antibody cocktail for mononuclear myeloid cells and activation markers: CD45 APC-Cy7 (clone 30-F11, BioLegend), CD11b PE-CF594 (clone M1/70, BD Biosciences), CD11c PE-Cy7 (clone N418, eBioscience), I-A/I-E BV421 (MHC-II clone M5/114.15.2, BD Biosciences), CD86 PerCP/Cy5.5 (clone GL-1, BioLegend), Ly6C AF647 (clone ER-MP20, Serotec), Ly6G PE (clone 1A8, BD Pharmingen). T cells were characterized using the following antibody mix: CD45 eFluor 450 (clone 30-F11, eBioscience), CD3e PE-Cy7 (clone 145-2C11, BioLegend, CD8a APC (clone 53-6.7, eBioscience), CD4 APC-Cy7 (clone RM4-5, BioLegend), CD44 PerCP (clone IM7, eBioscience). Samples were acquired on an LSRII (BD Biosciences) at the Cell Analysis Core, UTSA. Data analysis was performed using FlowJo v10. For cell sorting, brain cell suspensions (pooled 3 mice per sample) were stained with antibodies against CD45 and CD11b and after gating for single cells the CD45^LoCD11b⁺ population was separated in a FACS ARIA-II (BD Biosciences).