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.