2.5. Cell adhesion assays
Cell adhesion activity was measured using the crystal violet assay. 96 well plates were coated with 1noml/mL of EGF, RHC and RHC/EGF overnight at 4℃. After washing with phosphate-buffered saline (PBS), the wells were blocked with 1% (w/v) bovine serum albumin for 30 min. NIH/3T3 cells were seeded in the same plates at 6 ×103 cells per well in FBS-free medium. After 2h, cells were washed twice with PBS and fixed with Methanol solution for 20 min at room temperature. Fixed cells were stained with 1% (w/v) crystal violet (Solarbio, China) for 30min at 37℃. Stained cells were then gently washed three times with PBS. Cell images were captured by MF53 microscope (Mshot, Guangzhou, China). The experiment was performed in quintuplicate, and the number of adherent cells was calculated from the images captured at five positions per well to determine the average. For cell lysis, 1% SDS solution was added. The absorbance was measured using a microplate reader at 570nm.