Ovarian Cancer Cell Clonogenic Assay

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)].