Ccr7 alexa 647

For extracellular staining 3×10⁵ cells were blocked with an unspecific IgG antibody 1:100 diluted in PBS 1X supplemented with 3% FBS for 15 min and then incubated for 30 min with mouse monoclonal anti-human CXCR3-PE-Cy7 (1:50, Clone: 1C6/CXCR3, ref. 560831), CXCR4-PE-Cy7 (1:50, Clone: 12G5, ref. 560669), CXCR7-APC (1:50, Clone: 10D1-J16, ref. 391406), CCR7-Alexa 647 (1:50, Clone: 150503, ref. 560816), CD44-PE (1:50, Clone: G44-26, 555479) (all antibodies were from BD Biosciences; except for anti-CXCR7 that was from BioLegend). Finally, cells were incubated with 7AAD (7-amino-actinomycin, BD Biosciences, ref. 559925). For intracellular staining cells were blocked with an unspecific IgG antibody 1:100, diluted in PBS 1X supplemented with 3% FBS, then cells were washed with Phosflow Perm/Wash Buffer I (1X) (PWB 1X; BD Biosciences, ref. 557885), and fixed and permeabilized using Cytofix/Cytoperm solution (BD Biosciences, ref. 554722). To block intracellular Fc receptors the cells were again incubated with the unspecific IgG antibody, and then incubated for 1 h with mouse monoclonal anti-human Sox-2-Alexa 488 (Clone: 245610, BD Biosciences, ref. 560301). All acquisitions were performed on a FACSAria flow cytometer (Becton Dickinson). Analysis of flow cytometry data was performed on viable 7-AAD negative cells (except for Sox-2 staining) using FlowJo V10 software (Tree Star, Inc.).