Establishment of a Standardized Assay System of Fibronectin Activity Using Fibronectin-Mediated Cell Adhesion

• Published in: Biological & pharmaceutical bulletin Vol. 20; no. 12; pp. 1219 - 1223
• Main Authors: SHIMIZU, M, MOON, M, SHIRONO, C, MINAKUCHI, K, NISHIMURA, T, KOGA, J, NISHIDA, T
• Format: Journal Article
• Language: English
• Published: Tokyo The Pharmaceutical Society of Japan 01.12.1997; Maruzen; Japan Science and Technology Agency
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Arg-Gly-Asp (RGD); baby hamster kidney cell; Biological and medical sciences; Cell Adhesion - physiology; Cell Line; cell-binding domain; Cricetinae; Culture Media; fibronectin; Fibronectins - analysis; Fibronectins - isolation & purification; Fibronectins - physiology; Fundamental and applied biological sciences. Psychology; Glycoproteins; Humans; neutral red; Polystyrenes; Proteins; Receptors, Fibronectin - metabolism; Reference Standards; Serum Albumin, Bovine; Human origin; Cell culture; Rodentia; Cell cell interaction; Glycoproteins; Adhesion; In vitro; Biological activity; Fibronectin; Protein; Kidney; Well testing; Vertebrata; Biological fixation; Mammalia; Investigation method; Animal; Hamster
• ISSN: 0918-6158; 1347-5215
• DOI: 10.1248/bpb.20.1219

An in vitro assay of fibronectin (FN) was established based on the adhesion of baby hamster kidney (BHK) cells through the cell-binding domain of FN.Each well of a microtiter plate was coated with samples or various concentrations of standard FN. Bovine serum albumin was further coated to prevent the non-specific adhesion of the cells. Various numbers of BHK cells were plated and incubated. After washing out the non-attached cells, the number of attached cells was measured using neutral red (NR)-staining.The conditions for the assay were optimal when 1×105 cells/well were plated and incubated for 90 min. The linear relationship between the concentration of FN coated and the absorbance of NR was observed in the range of 0.1-1.0 μg/ml of FN. The inhibition of cell binding by the peptides containing an Arg-Gly-Asp (RGD) sequence demonstrated that this assay system depended on FN-mediated cell adhesion through the major cell-binding domain.