To isolate pools of cells expressing different levels of E-cadherin, a p120 siRNA-infected A431 cell population was sorted by FACS® as follows: cells were dissociated with GIBCO BRL cell dissociation buffer (enzyme free, PBS based) at 37°C for 45 min. Single-cell suspensions were enhanced by repeated pipetting, washed in PBS containing 1% serum, and then labeled with E-cadherin mAb HECD1 (10 μg for 5 × 106 cells in 1 ml), followed by washing and then additional labeling with the secondary antibody Alexa® 488–conjugated goat anti–mouse IgG (1/1,000 dilution in 1 ml; Molecular Probes, Inc.). After washing, cells were labeled with 7AAD (Molecular Probes, Inc.) to discriminate dead cells, and subjected to FACS® using a FACStar PLUS™ cell sorter (Becton Dickinson). All procedures were performed at 4°C to prevent E-cadherin endocytosis. Four gates were set based on preliminary experiments designed to separate cells into four categories of cells expressing high to low levels of E-cadherin. The resulting pools were expanded and then analyzed by Western blotting for p120 and E-cadherin levels.
