To determine if the goat polyclonal R&D EpCAM antibody could capture feline tumor cells under static conditions, we used a modification of our previously described assay (32). For this assay, we compared the feline oropharyngeal squamous cell carcinoma cell lines (SCCF-2, SCCF-3), with the MCF-7 human breast carcinoma and the C10 feline injection site sarcoma cell lines as positive and negative cell controls, respectively. In each experiment, duplicate coverslips were coated for 1 h at 37°C with the goat polyclonal R&D EpCAM antibody (1:50 final dilution, 4 μg/ml), using equivalent concentrations of goat γ-globulin as a negative control and fibronectin (20 mg/ml, EMD Millipore, Burlington, MA) as an integrin-mediated binding control. The rabbit monoclonal SB EpCAM antibody (1:50) was used as a positive antibody-binding control for the human MCF-7 cell line. After incubation, coverslips were washed three times with sterile PBS, and then blocked by incubation with 1% PBS-BSA for 30 min at 37°C. Coverslips were then seeded with 5 × 104 cells and incubated at 37°C for 1 h to allow for attachment. The coverslips were subsequently washed before fixing the cells with 4% paraformaldehyde. After additional washes, the coverslips with adherent cells were mounted to glass slides with Prolong™ Gold antifade mounting media, which includes 4′-,6′- diamidino-2-phenylindole for nuclear staining (Thermo Fisher Scientific). Ten random fields were then captured using an Axio Imager M1 microscope (Zeiss, Thornwood, NY, USA). A blinded investigator then reviewed the captured saved images and counted DAPI-stained nuclei of adherent cells. Each cell line was tested in at least two separate experiments.

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