Cd86 percp cy5.5 clone gl 1

Mouse macrophages (RAW264.7), mouse melanoma (B16F10), and mouse renal adenocarcinoma (RENCA) cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Murine breast cancer cells (E0771) were obtained from CH3 BioSystems (Buffalo, NY). All cell lines were routinely grown in high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, Gaithersburg, MD), and an antibiotic/antimycotic solution at final concentrations of 100 U/ml penicillin, 100 μg/ml streptomycin and 0.250 μg/ml amphotericin B (Sigma-Aldrich, St. Louis, MO). Cells were maintained in T-25 tissue culture flasks (Techno Plastic Products AG, Trasadingen, Switzerland) in a standard humidified incubator with 5% CO₂ balanced-air at 37°C. Prior to experimentation, RAW264.7 cells were serum deprived. Fluorescence-conjugated antibodies purchased from BioLegend (San Diego, CA) were employed to identify macrophages (CD45-FITC, clone30-F11) and to determine their phenotype (CD86-PerCP/Cy5.5 -clone GL-1- for M1 phenotype and CD206-APC -clone C08C02- for M2 phenotype) by fluorescence-activated cell sorting (FACS).

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).