Flow cytometry analysis of integrin LFA-1 activation

The percentage of Ba/F3 cells expressing C3G-mEGFP, WT or mutants, or mEGFP alone with activated integrin LFA-1 was determined by flow cytometry using the antibody mAb24 (ab13219, Abcam), which recognizes the activated conformation of this integrin. Ba/F3 cells were washed in ice-cold PBS containing 3% BSA and were stained with mAb24 (1/100 dilution; 60 min, 4°C). Cells were washed and stained with Cy5-conjugated goat anti-mouse IgG secondary antibody (1/200 dilution; 30 min, 4°C) (Jackson ImmunoResearch). Cells were washed; resuspended in cold PBS, 3% BSA, and 1% sodium azide; and analyzed using an Accuri C6 flow cytometer (BD Biosciences). Cells processed in a similar manner but without the primary antibody were used as a control of unspecific labeling by the secondary antibody. Data were analyzed using the BD Accuri C6 software.