Allophycocyanin apc beads

Hydrogel discs were placed in ultralow binding polystyrene well plates (Corning) to ensure cells attach to hydrogels and not the plate surface. Hydrogels were washed with Hank’s buffered salt solution (HBSS, Thermo) for at least 3 days before seeding cells. Cells were seeded at a low density (25 cells/mm²). After seeding, hydrogels were washed thoroughly to remove unattached cells. Drug treatments of 3 μM MnCl₂ (Fisher Scientific), 200 nM cilengitide (Cayman) and 5 μM CK869 (Cayman) were applied during and after cell adhesion to substrates. To evaluate the number of live cells seeded on the substrate, cells were detached by incubating with Accutase Cell Detachment Solution (Innovative Cell Technologies, Inc.) for 10 min at 37 °C. Cells were then washed by centrifugation and directly added to HBSS containing calcein AM (1:2000; Biotium), ethidium bromide (1:2000; Thermo Fisher Scientific), and a predefined number of allophycocyanin (APC) beads (BD). After incubation at room temperature for 10 min, the samples were analyzed for live and dead cell number by flow cytometry.