Versene 0.48 mm edta

For FACS analysis, cells were harvested with versene/0.48 mM EDTA (Invitrogen, UK), washed with PBS supplemented with 0.5% FBS and re-suspended in the stain buffer (2 mM EDTA and 0.5% bovine albumin in PBS). Single cell suspension was labeled with APC-conjugated P-cadherin, GLUT1 and CAIX antibodies. Cells transfected with the control siRNA and with CDH3 siRNA were doubled stained either with P-cadherin and GLUT1 or CAIX antibodies. A live-dead stain (Invitrogen, UK) and the primary and secondary antibodies were incubated at 4°C, in the dark, for 15 minutes. Secondary Alexafluor-488-conjugated goat anti-rabbit IgG (Invitrogen, UK) was used in a 1:100 dilution. The labeled cells were then washed in the stain buffer and analyzed on a FACS Canto-II (BD Biosciences, USA).
For the sorting experiments, the subpopulations of SUM149 and BT20 breast cancer cells were selected according to P-cadherin expression (highest and lowest 20% expressing cells). Cells were sorted using BD Influx or FACS ARIA-II (BD Biosciences) and collected into 10% Hanks buffered solution (Invitrogen, UK). The purity of sorted populations was 80-95%. In addition, a further sample was also collected of cells passed through the laser under pressure, but not sorted, to act as a control for the effect of the pressure on the cells.

To determine the expression of CD44, CD24, and CD49f on the cell surface of 231, 231.Br, and 231.Br.HER2 cells, we performed flow cytometry analysis. Cells were washed twice with phosphatase buffered saline (PBS) solution and then harvested with versene/0.48 mM EDTA (Invitrogen). Detached cells were washed with PBS supplemented with 0.5% FBS (stain buffer), centrifuged at 1200 rpm for 5 min, and resuspended in stain buffer. A single-cell suspension was incubated with APC-conjugated anti-CD44 (1:20) and PE-conjugated anti-CD24 (1:10), or with PerCP-Cy7-conjugated anti-CD49f (1:20). All antibodies were purchased from BD Biosciences (Temse, Belgium). Primary antibodies or the respective isotype controls (BD Biosciences) were incubated at 4 °C in the dark for 20 min. A cell viability marker was included (1:100 dilution, violet fluorescent reactive dye, Invitrogen) to discriminate dead cells. Cells were analyzed using a BD FACS Canto-II flow cytometer (BD Biosciences, Temse, Belgium).