Facscan canto 2

Cells previously cultured in 75 cm² flasks were detached with versene (Gibco), resuspended in fresh medium, and centrifuged at 300g for 5 min. Cells (1 × 10⁶ cells/ml) were washed two times in PBS and 1% bovine serum albumin (BSA) (Sigma–Aldrich) solution, and each wash followed by centrifugation at 300g for 5 min. The cell pellet was resuspended and incubated 1 h at room temperature (RT) with the primary antibody 10E4 (Table 2) in PBS and then washed again with PBS 1% BSA solution and incubated with APC fluorochrome-conjugate antimouse IgM. For negative controls, cell pellets were incubated with APC fluorochrome-conjugate antimouse IgM only. Finally, cells were washed again and resuspended in PBS 1% BSA solution and filtered for analysis. Data were acquired using FACscan Canto II and analyzed with the FlowJo software (v10; BD Biosciences). Mean intensity of fluorescence values measured for the KO clones were normalized to the mean intensity of fluorescence values of WT, which were defined as a unit value. Three independent experiments were analyzed.

Cell surface expression of αvβ3 integrin and SDC4 was analyzed via FACscanCantoII (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, cells were detached using accutase (Sigma Aldrich, Saint Louis, USA), washed in a PBS1X-BSA 0.3% solution and kept on ice for the remaining procedure. Cells (5 × 10⁵ cells/ml) were then incubated 1 h at 4° C with primary anti-αvβ3 or anti-SDC4 antibodies (10 µg/ml), washed with PBS1X-BSA0.3%, and incubated with FITC flurochrome-conjugated anti-mouse IgG or anti-rat IgG (1:2000 dilution). Cells were finally washed and suspended in PBS1X-BSA0.3% for analysis.