Ortho fluorescence microscope

Transwell inserts (8 μm) were incubated in DMEM supplemented with 1% fetal bovine serum overnight. Approximately 3 × 10⁴ B16F10, LLC or MC38 cells were seeded in the lower chambers, and chambers containing only DMEM served as controls. After 10 h of incubation for adherence, the medium was refreshed with 700 μL of DMEM supplemented with 10% fetal bovine serum and 50 μg/mL gentamicin. RAW264.7, RAW264.7(VNP), PEMΦ, and PEMΦ (VNP) cells were prepared, and the concentration was adjusted to 1 × 10⁶ cells/mL using DMEM. One hundred microliters of the cell suspension was transferred to the Transwell insert and incubated at 37 °C for 16 h. Then, the Transwell inserts were removed from the plate, and the remaining cells that had not migrated from the top of the membrane were removed using a cotton-tipped applicator. After being washed 2–3 times with PBS, the Transwell inserts were placed in 4% paraformaldehyde for 20–30 min and then stained with crystal violet (KeyGen BioTech, KGA229, Nanjing, China) for 30 min at room temperature. After being washed with PBS 2–3 times, the Transwell inserts were photographed under an ortho-fluorescence microscope (Carl Zeiss).