2.8. Anoikis assay (FACS and caspase 3/7 activity)

For FACS, cells were seeded in ULA 10‐cm dish (#3263; Corning) at a density of 500 000 cells per dish. After incubated at 37 °C, 5% CO₂ for 48–72 h, cells were collected, trypsinized to get single cell suspension, and stained with propidium iodide (PI) and Annexin V (#V13242; Sigma) for 15 min at room temperature in dark. LSRII FACS analyzer was used to do the FACS with proper gating.

For the caspase 3/7 activity assay, cells were seeded in ULA 96‐well plates (#7007; Corning) at a density of 10 000 (OV17R, OVCA429, and OV7 clones) or 5000 (CH1 clones) cells per well. After 72‐h or 96‐h incubation, 20 μL of CellTiter‐Fluor reagent (for cell viability, #TB371; Promega (Madison, WI, USA)) was added to all the wells and the fluorescence was measured using a Tecan plate reader (Tecan, Männedorf, Switzerland; infinite 200) after 1‐h incubation at 37 °C. One hundred microlitee of caspase‐Glo 3/7 reagent (#TB323; Promega) was then added to all the wells, and the luminescence was measured after 1–2 h incubation at room temperature. The caspase 3/7 activities were divided by the cell viabilities and then normalized to their respective controls.