2.13. Anoikis Assay

For fluorescence microscopy, 96-well plates were pre-coated with polyhema and 1 × 10⁵ PC3 cells were seeded and treated with varying concentrations of NCX4040 for 24 hr. Afterwards, Calcein green stain to detect live cells and ethidium homedimer (EtHD) for dead cells was added to wells and incubated for 30 min at 37°C, images were captured using fluorescence microscope. Image J was used to quantify the fluorescence intensity. In addition, MTT was added and incubated for overnight to check cell viability. For flow cytometry, 1X10⁵ PC3 cells were seeded onto polyhema pre-coated 6-well plate and incubated for overnight. Following, cells were treated with varying concentrations of NCX4040 for 24 hrs. The cells were collected, washed with 1XPBS and stained with Calcein green and ethidium homodimer for 30 min and analyzed by flow cytometry. Data were analyzed using Flow Jo software.