Anoikis assay and caspase 3 activity determination

Poly-2-hydroxyethyl methacrylate (poly-HEMA, Sigma-Aldrich) was prepared by dissolving it in 95% ethanol (v/v) to a concentration of 12 mg/mL and subsequently added to cell culture wells at a density of 5 mg/cm². Cells were cultured for 24 h using poly (HEMA)-treated (suspended) dishes. Then, anoikis rate of cells was determined by the FITC Annexin V/7-AAD Apoptosis Kit (BD Biosciences, San Jose, CA, USA) and analyzed by BD FACS Calibur system, and data were analyzed with FlowJo software (San Carlos, CA, USA). Caspase 3 activity were tested by Caspase 3 Activity Assay Kit obtained from Beyotime according to the manusfactuer's protocol. Cell lysates were prepared by incubating 2×10⁶ cells/mL in extraction buffer for 30 min on ice. Lysates were centrifuged at 13,000×g for 15 min, and the supernatants were collected. The protein concentrations were determined by BCA protein assay (Beyotime). Cellular extracts (40 μg) were then incubated in a 96-well microtitre plate with 20 ng Ac-DEVD-pNA for 2 h at 37 °C. Caspase 3 activity was measured by cleavage of the Ac-DEVD-pNA or Ac-LEVD-pNA substrate to pNA, the absorbance of which was measured by Varioskan Flash (Thermo Fisher Scientific) at 405 nm. Relative caspase activity was calculated as a ratio of emission of treated cells to untreated cells.