Apc conjugated anti epcam

Cells were grown in pWIT or svWIT medium as indicated in the figures, detached
using 0.5% Trypsin-EDTA (Invitrogen), pelleted, washed and counted.
10⁵ cells were incubated for 20 minutes with the
following antibodies: allophycocyanin (APC)-conjugated anti-EpCAM; phycoerythrin
(PE)-conjugated anti-CD49f; PE-conjugated anti-CD10 (BD Biosciences). Non-specific
PE-IgG2a and APC-IgG1 (BD Biosciences) were used as control antibodies. The gates
marking the quadrants in the figures were set to exclude cells labeled by the
control antibodies. Cells were washed three times then analyzed on a FACSCalibur
flow cytometer (Becton Dickinson, San Jose, CA, USA) using CellQuest Pro
software.

For flow cytometry analysis, cells were cultured mono/co-culture spheroids, and then collected and trypsinized to obtain a single-cell suspension. Briefly, cells were washed in FACS buffer (PBS supplemented with 2% FBS) and, stained according to the manufacturer’s instructions in 100 µL FACS buffer supplemented with APC-conjugated anti-EPCAM (1:50, BD Biosciences, San Jose, CA, USA), PerCP-Cy 5.5-conjugated anti-CD56 (1:100, BDBiosciences, USA), PE-conjugated anti-CD107a (1:100, BD, Biosciences, USA) and APC conjugated anti α-SMA (1:2000, R&D Biotechne, Minneapolis, MN, USA). Cells were kept in the dark at RT for 20 min. Live/Dead cell analysis, Annexin-V-FITC (1:100, Cell Signaling Technology, Danvers, MA, USA) and Propidium Iodide-PE (1:10, Cell Signaling Technology, USA) were used. Stained cells were washed twice in DPBS to remove unbound antibodies, resuspended in 200 µL FACS buffer and analyzed in BD FACSCalibur Flow Cytometer (BD Biosciences). An unstained negative control was run to establish the fluorescence gates. First, the cells were gated in an FSC-A (forward scatter) and SSC-A (side scatter) dot plot to eliminate doublets. EPCAM (+) cells were chosen to analyze dead/live ratio in cancer cells. In order to identify CD107a (+) NK-92 cells, the CD56 (+) cell population was gated. All data were then analyzed using FlowJo X software Version 9.