Giemsa staining solution

Ovarian cancer cells at the logarithmic growth phase were collected, digested by 0.25% trypsin, triturated, counted, and adjusted to 1 × 10⁶ cells/mL. Each group of cells was inoculated with a gradient density of 50, 100, and 200 cells, respectively, in a 10-cm dish that was cultured in a 37 °C 5% CO₂ incubator for 2–3 weeks. The culture was terminated when the clone was visible in the culture dish. Then, the cells were fixed by 4% paraformaldehyde (5 mL, Invitrogen) for 15 min and stained by Giemsa staining solution (5 mL, Invitrogen) for 10–30 min. Finally, the cells were washed slowly with PBS and dried in air. The plate was observed under the inverted microscope (DMi8-M, Leica, Wetzlar, Germany). The number of cell clones was recorded and the clone-formation rate was calculated as the number of formed clones / the number of inoculated cells [57 (link), 58 (link)].

A total of 5 × 10⁴ cells were seeded on BD Falcon Cell Culture Inserts with or without a thin layer of MATRIGEL Basement Membrane Matrix. The inserts were then placed on 24‐well plates containing complete serum as chemo‐attractant. After incubation in serum‐free medium for appropriate time (16 h for H1299 migration assay, 24 h for H1299 invasion assay, 38 h for HCC827 migration assay, and 48 h for HCC827 invasion assay), inserts were washed with PBS, fixed with 4% formalin (Sigma), and stained with Giemsa staining solution (Invitrogen). The unmigrated cells on the surface of the membrane were removed using cotton swabs. Images were taken and analyzed with imagej (National Institutes of Health, Bethesda, MD, USA) to acquire cell numbers.