Fig. 4

The canine squamous cell carcinoma epithelial cell line CSCAG891 binds rapidly to LTC. A canine squamous cell carcinoma cell line, CSCAG891, was established from an excised canine carcinoma tumor. Cells were grown in 6-well plates to approximately 90% confluence in DMEM 10% FBS. Triplicate cultures of CSCAG891 cells were treated with either nothing (a) or 10 μl of TopFluor®-labeled LTCs for 5 min followed by washing 3X in medium (b). For microscopy, images of CSCAG891 cells either untreated or treated with 10 μl of TopFluor®-labeled (green arrow) CSCAG cells were subsequently stained with the cross-reactive human epithelial cell marker EpCAM (orange arrows) and nuclei stained with DAPI (blue arrows). For analysis of adherence of LTC to CSAG891 cells, cells were treated with LTC and removed from wells by treatment with trypsin, filtered and assayed by flow cytometric analysis for TopFluor®- stained cells over a period of 50 min, (c). Measured green channel (FL1) gMFI for triplicate samples was compared to unstained controls over each time point and differences were analyzed by ANOVA with a Geisser-Greenhouse’s epsilon value of 0.2802. (****, P ≤ 0.001)