Matrix glue

The invasiveness of cells was evaluated using the transwell assay (BD Biosciences, Bedford, MA, USA). The matrix glue (BD Biosciences) was diluted 6:1 in serum-free medium, evenly spread on the membrane in the upper layer of the transwell and placed in the incubator at 37 °C for 2 h to solidify. The cells were then diluted in a serum-free culture medium, added to the lower transwell chamber (200 μL of cell suspension; 8 × 10⁴ cells) along with 600 μL of culture medium with 10 % FBS, and incubated for 24 h. The cells were then fixed with 4 % paraformaldehyde (20 min), washed with PBS, and stained with a crystal violet solution for 15 min. Subsequently, the upper layer cells were air-dried and imaged in an inverted fluorescence microscope. The Image J software was used to analyze and quantify cell invasiveness, with cells stained with crystal violet defined as positive. The average number of stained cells in six random fields, in each compartment, was calculated.

A density of 2×10⁴ cells per well in 200 μL of serum-free medium was used to seed the cells in the upper chamber of a transwell plate. The upper chamber was either coated or uncoated with matrix glue (BD Biosciences, USA). The lower compartment was loaded with 700 μL of complete medium supplemented with 10% serum. After 36 hours of incubation, the cells were fixed, stained, and counted by microscopy. Images were captured for analysis.

Transwell chambers were placed on a 24-well plate, and osteosarcoma cells in the logarithmic growth phase were digested with trypsin and washed with PBS and serum-free medium. 1∗10⁵ cells and serum-free medium were inoculated in the upper compartment of each well. DMEM medium containing 10% fetal bovine serum was added to the lower compartment. For cell invasion assay, 50 μl diluted matrix glue (BD Biosciences, Franklin Lakes, NJ) was added to the upper chamber and incubated at 37°C for 4-5 hours; the following operation is the same as above. The cells were cultured in a 37°C incubator for 22 hours in the migration experiment and 24 hours in the invasion experiment. The cells that have migrated or invaded are fixed, stained, and counted at a specified time.

Transwell experiments were divided into transwell migration and transwell invasion experiments. The basic operations were as follows: transwell cells were placed into a 24-well culture plate, the chamber is referred to herein as the superior chamber and the culture plate is referred to as the lower compartment. The cells were then digested in a serum-free medium, following which the cell density was adjusted to 1 × 10⁶ cells/mL and the sample was inoculated in the upper chamber. Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS was then added to the lower chamber. Transwell invasion assays were performed by precoating the upper membrane with 40 µL of matrix glue (BD Biosciences, USA); the cells were fixed with 4% paraformaldehyde and were washed with phosphate buffer solution (PBS) after 24 h. Then, they were stained with 0.1% crystal violet (Solarbio, China) and representative images were observed at × 100 and × 200 magnification with an optical microscope (Olympus, Tokyo, Japan). Five non-repeating fields per chamber were selected for photography and counted.