Ratcol rat tail collagen

The Vybrant Cell Adhesion Assay Kit (Thermo Fisher) was used to measure EC adhesion to microplate wells coated with Type I collagen solution (RatCol Rat Tail Collagen, Advanced BioMatrix, at a working concentration of 35 μg/ml. Microplate wells were incubated at room temperature, covered, for 1-2 hours with the diluted Rat Tail collagen added to the well surface. Remaining material was aspirated, and coated surfaces were rinsed with PBS. Cells were resuspended in serum-free medium at 5 x 10⁶ cells/ml, and then calcein AM stock solution was added to cell suspensions at a concentration of 5 μM. Cells were incubated for 30 minutes at 37°C, washed twice with serum-free medium, and then 100 μL of the calcein-labeled cell suspension was added to the coated microplate wells. Cells were incubated for 120 minutes at 37°C, followed by removal of nonadherent calcein-labeled cells by 5 washes. Fluorescence was then measured with a Varioskan LUX Multimode Microplate Reader (Thermo Fisher) at an Ex/Em of 494 nm/517 nm. The percentage of adhesion was calculated by dividing background-subtracted fluorescence of adherent cells by the total corrected fluorescence of cells added to each microplate well, multiplied by 100%.