Cd45 pe

Freshly thawed PBMCs were resuspended in serum-free, antibiotic-free RPMI 1640 media. Cells were distributed (0.5 x 10⁶cells in 100 μL per tube) to Falcon polystyrene round-bottom tubes (12 x 75 mm; Corning Incorporated, Durham, NC) and treated with IL-6 or IFN-γ (100 ng/mL) (R&D Systems, Minneapolis, MN) for 15 min at 37°C before subjecting them to phospho-specific flow cytometric analysis as described previously [7 (link)]. Briefly, after stimulation, cells were fixed by incubating in 2% PFA (BD Cytofix Fixation Buffer; BD Biosciences) for 10 min at 37°C and pelleted. They were then permeabilized by resuspending with vigorous vortexing in 300 μL ice-cold methanol. Cells were washed in staining media. Fluorophore-specific MAbs were added and incubated for 30 min at RT. The following markers were analyzed: CD3-PE/Cy7, CD4-APC/Cy7, CD45RO-PerCP/Cy5.5, CD33-APC, STAT1 (pY701)-AF488, STAT3 (pY705)-AF488, (BD Biosciences, San Diego, CA); and CD45-PE (R&D Systems, Minneapolis, MN). The cells were washed with staining media and pelleted. Finally, the samples were resuspended in 250 μL of staining media and analyzed.

Following anesthesia of animals by overdose administration of a ketamine (Merial)/xylazine (Bayer) cocktail by intraperitoneal injection, the ipsilateral mouse striatum was dissected, passed through 70 μm nylon cell strainers (BD Falcon) and single-cell suspensions were generated. 2 × 10⁵ total cells were stained with fluorescently-labeled antibodies against mouse CD45-FITC (Clone:30-F11) (1:100), CD45-PE (Clone:30-F11) (1:100), CD45-PE-Cyanine 5 (Clone:30-F11) (1:100), CD11b-PE-Cyanine 5 (Clone:M1/70) (1:100), CD11b-PE-Cyanine 7 (Clone:M1/70) (1:100), Arginase-1 (Arg-1)-FITC (R&D Systems) (1:100), nitric oxide synthase 2 (NOS-2)-PE Cyanine 7 (Clone: CXNFT) (1:100), MHCII- I-Ab-FITC (Clone:AF6–120.1) (1:100), CD86-PE (Clone:GL1) (1:100) (eBioscience) for 30 minutes. For intracellular IL-10 staining, 10⁶ cells were stained with fluorescently-labeled antibody against mouse IL-10-PE (Clone: JES5–16E3) (1:100), according to the manufacturer’s instructions (BD Biosciences). FACS acquisition was performed with the cytometer Cytomics FC500 (Beckman Coulter) and the data were analyzed using the FlowJo software 8.7 (Tree Star, Inc., Ashland, OR).