Establishment of a standardized assay system of fibronectin activity using fibronectin-mediated cell adhesion.
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 x 10(5) 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 micro/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.
Shimizu
M
M
Development and Research Laboratories, JCR Pharmaceuticals Co., Ltd., Kobe, Hyogo, Japan.
Moon
M
M
Shirono
C
C
Minakuchi
K
K
Nishimura
T
T
Koga
J
J
Nishida
T
T
D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D002460:Cell Line; D006224:Cricetinae; D003470:Culture Media; D005353:Fibronectins / Q000032:analysis* / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D011137:Polystyrenes; D017469:Receptors, Fibronectin / Q000378:metabolism; D012015:Reference Standards; D012710:Serum Albumin, Bovine
D003470:Culture Media; D005353:Fibronectins; D011137:Polystyrenes; D017469:Receptors, Fibronectin; D012710:Serum Albumin, Bovine