Anti cxcr3 fitc

Cell surface markers expression was analyzed by flow cytometry using a Navios cytometer and data evaluated using Kaluza software (Beckman Coulter, USA).
A surface staining protocol was performed to analyze membrane antigen expression in lymphocytes subpopulations. Briefly, 100 µL of peripheral fresh whole blood were incubated 20 min at room temperature (25°C) with different fluorochrome-conjugated monoclonal antibodies combinations. Red blood cells were lysed, white cells fixed using TQ-Prep System (Beckman Coulter), and 100 µl of fluorospheres Flow-Count™ (Fluorospheres, Beckman Coulter) were added to calculate cell concentration, previous to flow cytometric analysis.
Combinations of the following antibodies were used: anti-CD45-FITC, anti-CD4-PE, anti-CD8-ECD, anti-CD3-PCy5, anti-CD56-PCy5, anti-CD3-PCy7, and anti-CD45RA-ECD (all from Coulter Immunotech, France) to evaluate T and NK cells; anti-CD19-ECD, anti-CD27-PCy7, anti-CD21-FITC, anti-CD24-PCy5, anti-CD38-PCy7 (all from Coulter Immunotech, France) and anti-IgD-FITC (Dako, Spain) to study B cells; anti-CD4-PCy5, anti-CD45RA-ECD (from Coulter Immunotech, France), anti-CXCR5-PE (R&D Systems, Spain), anti-CXCR3-FITC and anti-CCR6-PCy7 (Biolegend, USA) to evaluate Th and cTfh subpopulations.

The lymphocytes from the spinal cord tissues were isolated by Percoll (GE Healthcare, Chicago, IL, USA) density-gradient centrifugation and analyzed by flow cytometry. Cells were stained with fluorochrome-conjugated monoclonal antibodies; mouse anti-CD45-Pacific blue (1:1000 diluted), anti-CD4-PE-Cy7 (1:1000 diluted), anti-CD8-PerCP-Cy5.5 (1:1000 diluted), anti-CD25-PE (1:1000 diluted), anti-CD69-FITC (1:1000 diluted), anti-CD44-APC-Cy7 (1:1000 diluted), anti-CXCR3-FITC (1:200 diluted), anti-CCR6-PE-Cy7 (1:200 diluted, BioLegend, San Diego, CA, USA), anti-IFNγ-FITC (1:100 diluted), anti-IL-17A-PE (1:200 diluted), and anti-FOXP3-APC (1:400 diluted, eBioscience, San Diego, CA, USA) and anti-T-bet-PE (3 µl per sample, BD, Franklin Lakes, NJ, USA). Intracellular cytokine staining was performed using an Intracellular Fixation and Permeabilization kit according to the manufacturer's instructions (eBioscience, San Diego, CA, USA). The samples were run on a BD Canto II cytometer (BD Biosciences, San Jose, CA, USA) and the results were analyzed by FlowJo software version 10.1 (BD, Franklin Lakes, NJ, USA).

TILs were stained with anti-CD3 Brilliant violet 570, anti-CD4 Brilliant violet 510, anti-CXCR3 FITC (all from Biolegend, San Diego, CA) and anti-CD8a APC-Cy7 (BD Biosciences, Franklin Lakes, NJ). After 15 min, cells were washed in PBS-0.1% FBS, and analysed by flow cytometry. Differentiation and maturation marker analysis based on CD45RA and CCR7 expression was performed as described previously.¹⁷