24 well plate chamber insert

5× 10⁴ HCT116 cells was plated in an 8-μm, 24-well plate chamber insert (Corning Life Sciences, catalog no. 3422) with 100 μL serum-free medium at the top of the insert and DMEM medium (Gibco) containing 10 % FBS (500 μL) at the bottom of the insert. Cells were incubated for 24 h and fixed with 4% paraformaldehyde for 15 min. After washing with PBS, cells at the top of the insert were scraped with a cotton swab. Cells adherent to the bottom were stained with 0.5% crystal violet blue for 15 min and then washed with double-distilled H₂O. The positively stained cells were counted in eight random fields under the microscope, and the average value of eight fields was expressed. All assays were performed in triplicate.

Cell migration assays were performed as previously documented (Xiong et al., 2021 (link)). Briefly, to produce a wound, the monolayer cells on the 6-well plate were scraped in a straight line with pipette tips. The plate was then washed with PBS to remove detached cells. Photographs of the scratch were taken at indicated time points using the Nikon inverted microscope (Ti-S). Gap width was calculated using GraphPad Prism software. For the Transwell assay, 2.5 × 10⁴ cells in 100 μl serum-free medium were plated in a 24-well plate chamber insert (cat. 3422; Corning Life Sciences) with a medium containing 10% FBS at the bottom of the insert. The cells were incubated for 24 h and then fixed with 4% PFA for 20 min. After washing, the cells were stained with 0.5% crystal violet.

For cell migration, 1-2×10⁵ cells in 200 μl of serum-free medium were plated in an 8.0 mm, 24-well plate chamber insert (354578, Corning Life Sciences), with medium containing 10% FBS at the bottom of the insert. The cells were incubated for 24 h and then fixed with 4% PFA for 5 min. After washing 3 times with PBS, the cells were stained with 0.5% crystal violet blue for 5 min and then washed with double-distilled H₂O. Cells on the upper surface of the insert were removed with a cotton swab. The positively stained cells were examined under the microscope. For the cell invasion assay, Matrigel-coated chambers (354483, Corning Life Sciences) were used instead of the chamber inserts used in the migration assay.

The cell migration assay was performed as documented (Shi et al., 2019 (link)). For wound healing assay, to produce a wound, the monolayer cells in 6-well plate were scraped in a straight line with pipette tips. Plate was then washed with warm PBS to remove detached cells. Photographs of the scratch were taken at indicated time points using Nikon inverted microscope. For Transwell assay, 2.5 × 10⁴ cells in 100 µL serum-free medium were plated in 24-well plate chamber insert (Corning Life Sciences, catalog no. 3422), with medium containing 10% FBS at the bottom of the insert. Cells were incubated for 24 h and then fixed with 4% paraformaldehyde for 20 min. After washing, cells were stained with 0.5% crystal violet blue. The positively stained cells were examined under the light microscope.

Cell proliferation assay was performed as previously described (Xu et al., 2020 (link)). Briefly, the indicated cells were plated onto 12-well plates, and the cell numbers were subsequently counted each day using an automated cell analyzer Countstar (Shanghai Ruiyu Biotech Co., Shanghai, China, IC1000). Cell migration assay was performed as previously described (Xiong et al., 2021 (link)). For transwell assay, 1-2×104 cells in 100 l serum-free medium were plated in an 8.0-µm, 24-well plate chamber insert (Corning Life Sciences, catalog no. 3422), with a medium containing 10% FBS at the bottom of the insert. Cells were incubated for 24 h and then fixed with 4% paraformaldehyde for 20 min. After washing, cells were stained with 0.5% crystal violet-blue. The positively stained cells were examined under the microscope.

1 × 10⁵ cells were plated in an 8.0 mm, 24‐well plate chamber insert (Corning Life Sciences, catalog no. 3422) with serum free DMEM medium at the top of the insert and the same medium containing 20% FBS at the bottom of the insert. Cells were incubated for 24 h and fixed with 4% paraformaldehyde for 15 min. After washing with PBS, cells at the top of the insert were scraped with a cotton swab. Cells adherent to the bottom were stained with 0.5% crystal violet blue for 60 min and then washed with double‐distilled H₂O. The positively stained cells were examined under the microscope.