Pe conjugated anti cxcr3

Flow cytometry was performed with a FACS Canto II and FACS Aria II (BD Biosciences, Mountain View, CA, USA), and the obtained results were analysed using FlowJo software (Tree Star Software, San Carlos, CA, USA). LMNCs were stained using the following monoclonal antibodies (mAbs): fluorescein isothiocyanate (FITC)-conjugated anti-DX5, APC-conjugated anti-NK1.1, APC-conjugated anti-CD120a, APC-Cy7-conjugated anti-T cell receptor (TCR)-β, PE-conjugated anti-CD69, PE-conjugated anti-CXCR3, PE-conjugated anti-natural killer group 2, member D (NKG2D), PE-conjugated anti-NK1.1, PE-conjugated anti-CD120b (all from BD Pharmingen, San Diego, CA, USA), and PE-conjugated anti-TRAIL (BioLegend, San Diego, CA, USA). Proliferative potential was tested by intracellular staining using FITC-conjugated anti- Ki-67 (Miltenyi Biotec, Auburn, CA, USA) or isotype control. Nonspecific FcγR binding of labelled mAbs was blocked by anti-CD16/32 (2.4G2) (BD Pharmingen). Dead cells were excluded from the analysis by light-scatter and/or propidium iodide staining.

For preparation of CNS mononuclear cells, brains and spinal cords from MOG_35–55-immunized mice were excised and dissociated for 45 min at 37 °C by digestion with collagenase IV (2 mg/ml, Sigmal-Aldrich) and DNase I (100 μg/ml, Sigmal-Aldrich) in DMEM medium. Dispersed cells were passed through a 70 μm nylon mesh and collected by centrifugation. CNS mononuclear cells were isolated through a Percoll density gradient and collected from the interface fraction between 37% and 70% Percoll. After extensive washing, suspensions of cells were stained with FITC-labeled anti-CD4, PE-conjugated anti-CD11b, APC-conjugated anti-CD45, PE-Cy7-conjugated anti-MHC ΙΙ, PE-Cy7-conjugated anti-CD8, PE-conjugated anti-CXCR3, PE-conjugated anti-CCR5 (all from BD Pharmingen, San Diego, CA, USA). Isotype controls were used for determination of negative cells. The stained cells were analyzed on a FACSAria instrument (BD Bioscience, San Diego, CA, USA). For Th1, Th17 and Treg cell analysis, cells were stained with surface marker, permeabilized with the Intracellular Fixation and Permeabilization Buffer Set (eBioscience, San Diego, CA,USA), and then stained with anti IFN-γ, anti-IL17A, GM-CSF, and anti-Foxp3 antibodies. All these antibodies were purchased from eBioscience, unless marked otherwise.