Cell adhesion test and centrifugation assay

SMCs were mixed with Zn²⁺ solutions and seeded into a 24-well plate (Falcon, Corning, US). The final cell density was 50,000 cells/well. The cells were allowed to attach for 2 h. Then medium was removed and cells were washed by DPBS. The images of adhered cells were taken by microscope (EVOS FL Cell Imaging System, AMG, US) and the plate was sealed by self-sticking tape (Fisher Scientific, US). The plate was inversely put into the rotor and centrifuged at 1000 rpm for 5 min. Cells were washed by DPBS and fixed by 4% paraformaldehyde (ChemCruz, Santa Cruz Biotechnology, US). Images of attached cells were taken by microscope and analyzed by Image J (NIH, US). At least 10 different fields were used for calculating attached cell density and cell retention ratio.