Flow Cytometry Analysis of Integrin Expression

Cell surface expression of various integrin receptor subunits or SLC3A2/CD98hc was measured by standard flow cytometry as previously described [32 (link)]. Briefly, the cells (1 × 10⁶) were resuspended in the blocking buffer (DMEM supplemented with 2% BSA and 2% FCS) for 30 min on ice. The cells were then incubated with appropriate primary antibody: Chemicon (anti-integrin β1 MAB 1997 (clone MB1.2); anti-integrin α5 5H10-27 (clone MFR-5); anti-integrin α6 MAB 1378 (clone NKI-GoH3)); BD Pharmingen (anti-integrin β4 553745; anti-integrin αv 552,299 (clone RMV); anti-integrin β3 553343; anti-CD98hc H202-141); Invitrogen (anti-integrin β5 14-0497-82 (clone KN52); anti-integrin α2 14-5971-85 (clone DX5)) or matched isotype control diluted in labelling buffer (DMEM supplemented with 2% FCS) for 1 h on ice. Unbound antibodies were removed by washing twice with PBS, 2% FCS, and the cells were treated with an appropriate fluorescein isothiocyanate (FITC)-conjugated secondary antibody in a labelling buffer for 45 min on ice. The cells were washed again as above and stained with the viability dye 4′, 6′- diamidino-2-phenylindole (DAPI at 0.5 µg/mL) immediately prior to analysis on a BD FACS Canto II Flow Cytometer (BD Biosciences).