Celltracker orange cmtmr dye c2927

The CRC cell lines HCT116, SW480, and HT29 were labeled with CellTracker Orange CMTMR Dye C2927 (Thermo Fisher Scientific, Waltham, MA, USA). Cell suspensions were adjusted to 100 cells per 10 μL by serial dilution, and the actual number of cells in 10 μL cell suspension was directly counted under a fluorescence microscope BZ-X800 (KEYENCE, Osaka, Japan). With orange fluorescence as an indicator, 100 cells were seeded (in triplicate) on 12-well tissue-culture-treated polystyrene (TCPS) dishes (IWAKI, Shizuoka, Japan), or on dishes coated with 0.25% (w/v) PMEA in methanol solution and 200 μg/mL fibronectin. Cells were incubated in standard medium at 37 °C overnight, and then washed twice with PBS. The attached cells were counted under a fluorescence microscope (BZ-X800).

The HT29 and DLD-1 cells were labeled with CellTracker Orange CMTMR Dye C2927 (Thermo Fisher Scientific), and the cell suspension was adjusted to 500 cells per 50 μL by serial dilution. The actual number of cells in 10 μL cell suspension was directly counted under a fluorescence microscope (BZ-X800). Next, a cell suspension, containing approximately 500–700 cells, was mixed with 10 mL of venous peripheral blood from a healthy donor, and added to an OncoQuick tube (Greiner bio-one, Kremsmünster, Austria). After centrifugation at 1600× g for 20 min at 4 °C, the cells were washed once with PBS, washed again with advanced DMEM (Thermo Fisher Scientific), and then collected in 1 mL standard medium.
One 200-μL-aliquot of the 1 mL cell suspension was used to count the CRC cells marked with orange fluorescence under a fluorescence microscope. Another 200-μL-aliquot was transferred into a chamber slide II (IWAKI) coated with 0.25% (w/v) PMEA in methanol solution and 200 μg/mL fibronectin (in duplicate). These cells were incubated in standard medium at 37 °C overnight. The next day, immunocytochemistry for EpCAM was performed, and stained tumor cells were counted under a microscope in the light field.