CD24, CD44, and CXCR3B Flow Cytometry

Cells were harvested using enzyme-free cell dissociation solution (Millipore). Cells were fixed using ice cold 70% ethyl alcohol, blocked with 3% FBS and stained with CXCR3B antibody (Proteintech, Rosemont, IL, USA) followed by APC-conjugated goat anti-mouse polyclonal antibody (R&D Systems). After extensive washing, cells were stained with FITC-conjugated anti-human CD44 (BD Pharmingen, San Jose, CA, USA) and PE-conjugated anti-human CD44 (BD Pharmingen). Flow cytometry analysis was performed on BD FACSCanto II cytometer. Data analysis was performed with FCS Express 6 software. The target cells were gated on FSC/SSC plot to remove debris, followed by a singlet gate on FSC-H/FSCW plot. CXCR3B+percentage was calculated from the CD44⁺CD24⁻ population in the University of Maryland Greenebaum Comprehensive Cancer Center.