Visualix camera

HCT116 cells were seeded in six-well plates at a density of 1×10⁵ cells per well, incubated at 37°C overnight, and transfected with miR-NC or miR-1291 or DCLK1-siRNA at a final concentration of 50 nM. After 24 h of transfection, single cells were reseeded in 96-Well Clear Ultra Low Attachment Microplates (Corning, Inc.) at a density of 1,000 cells per well. The cells were cultured in DMEM/F-12 serum-free medium (Thermo Fisher Scientific, Inc.) supplemented with 20 ng/ml epithelial growth factor, 10 ng/ml basic fibroblast growth factor-2 (PeproTech, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were cultured in a humidified incubator at 37°C and 5% CO₂. Images were captured by a bright field light microscope (CKX53; Olympus Corporation) with Visualix camera (Visualix, Corporation). The number of spheres was counted manually on days 4 or 7 after reseeding.

Corning BioCoat Matrigel Invasion Chambers (pore size: 8.0 µm; Corning, Inc.; cat. no. 354480) were used. The upper chamber, which was pre-coated with Matrigel, was rehydrated with culture medium (RPMI-1640 or DMEM) for 2 h at 37°C in 5% CO₂ before seeding the cells. The medium (RPMI-1640 or DMEM) was then removed, and DLD-1, HT29 and HCT116 cells were seeded into the upper chambers at a density of 1-2×10⁵ cells per chamber with medium (RPMI-1640 or DMEM) containing 0.1% bovine serum albumin. Medium (RPMI-1640 or DMEM) containing 10% FBS were put in the lower wells. The cells were transfected with the miRNAs or antagomir or DCLK1-siRNA at a final concentration of 50 nM and incubated at 37°C after transfection. After incubation for 48, 72 and 96 h, cells passing through the Matrigel were fixed with 10% formalin for 1 h at room temperature and then stained with hematoxylin for 1 h at room temperature. To count cells passing through the Matrigel, images were captured using a bright field light microscope (CKX53; Olympus Corporation) with Visualix camera (Visualix, Corporation) at a magnification of ×200.

Ibidi culture inserts (8.4 width ×8.4 length ×5 mm height; Ibidi GmbH) with two 70 µl wells were put on the 24-well plates before cell seeding. DLD-1, HT29 and HCT116 cell suspensions (70 µl) were then added into each 70 µl well at a density of 2×10⁵, 2.5×10⁵ and 8×10⁵ cells per ml, respectively. The inserts were removed after 24 h to create gap with an area of 6×10⁵ pixel². After that, the miRNAs or antagomir or DCLK1-siRNA were transfected at a final concentration of 30 nM. To enhance gap closure, cells were cultured in the medium (RPMI-1640 or DMEM) supplemented with 10% FBS as previously described (50 (link),51 (link)). At 24 and 48 h after transfection, images were captured by a bright field light microscope (CKX53; Olympus Corporation) with Visualix camera (Visualix, Corporation) at a magnification of ×100. The areas of the gaps were measured using ImageJ 1.52v software (National Institutes of Health).