The acid-solubilized collagen stocks were diluted to 20 µg/mL in PBS and coated onto 96 well plates overnight at 4 °C. The well plates were rinsed with PBS and blocked with 1% BSA/PBS for 60 min at room temperature before rinsing with PBS. Prior to cell seeding, cells were suspended at 1 × 106/mL in media without serum before 350 µL aliquots were pipetted into Eppendorf tubes containing either 7 µL of anti-β1 integrin subunit antibody (Cat: MABT821, Merck KGaA, Darmstadt, Germany) at 500 µg/mL (10 µg/mL final), 1.75 µL of anti-αVβ3 integrin antibody (Cat: MAB1976, Merck KGaA, Darmstadt, Germany) at 1 mg/mL (5 µg/mL final), 7 µL of heparin sodium salt (Cat: H3149, Merck KGaA, Darmstadt, Germany) at 50 mg/mL in ultrapure water (1 mg/mL final) or combinations of all 3 treatments.
All of the binding inhibition reactions were incubated at 37 °C for 30 min before adding 50 µL/well (16,000/cm2) in 3 replicate wells for each collagen coating. Serial dilutions of the untreated cell stock were also seeded onto both collagen coatings in order to create a standard of known cell number for later quantification of relative adhesion for treated groups.
The cells were allowed to adhere for 2 h before washing with PBS and fixing in 4% PFA for 30 min. Wells were then washed three times in PBS and stained with 0.1% aqueous Crystal violet solution (Cat: V5265, Merck KGaA, Darmstadt, Germany) for 1 h. After washing away excess dye three times with PBS, cells were lysed with 100 µL 10% acetic acid for 15 min on an orbital shaker. The absorbance of the released dye was then measured at 575 nm using a microplate photometer.
All of the binding inhibition reactions were incubated at 37 °C for 30 min before adding 50 µL/well (16,000/cm2) in 3 replicate wells for each collagen coating. Serial dilutions of the untreated cell stock were also seeded onto both collagen coatings in order to create a standard of known cell number for later quantification of relative adhesion for treated groups.
The cells were allowed to adhere for 2 h before washing with PBS and fixing in 4% PFA for 30 min. Wells were then washed three times in PBS and stained with 0.1% aqueous Crystal violet solution (Cat: V5265, Merck KGaA, Darmstadt, Germany) for 1 h. After washing away excess dye three times with PBS, cells were lysed with 100 µL 10% acetic acid for 15 min on an orbital shaker. The absorbance of the released dye was then measured at 575 nm using a microplate photometer.
Free full text: Click here
