Fibronectin is a broad range natural cell adhesion factor. It is a 440-500 kDa dimeric glycoprotein consisting of two similar 220-250 kDa subunits linked by two disulfide bonds. It is found as a dimer in plasma and in multimeric form in the extracellular matrix and on cell surfaces. Its primary function is related to cell adhesion to the extracellular matrix which occurs via the Arg-Gly-Asp-Ser (RGDS) sequence of fibronectin with the appropriate transmembrane integrin receptor of the cells. Other domains of fibronectin are also involved with this adhesion process and may involve interaction with collagen, heparin and other cell surface glycosaminoglycans. The conformation and orientation of adsorbed fibronectin is also important and has an effect on cell spreading and strength of adhesion of endothelial cells.
This product is used as a thin coating on tissue-culture surfaces to promote attachment, spreading, and proliferation of a variety of cell types. The principal functions of fibronectin appear to be in cellular migration during wound healing and development, regulation of cell growth and differentiation, and haemostasis/thrombosis.
Fibronectin addition to serum free medium promotes cell adhesion. More significant effects are observed with BHK, CHO and other cell lines by coating of cultureware with fibronectin at 1-5 ug/cm².
Quality
- Purity ≥90% (SDS-PAGE)
- Tested for ability to promote attachment and spreading of BHK-21 cells
- Found negative for bacteria, fungi, and mycoplasma
Source: Human plasma.
NOTE: The source plasma was tested and found nonreactive for hepatitis B surface antigen (HBsAg) and negative for antibodies to human immunodeficiency virus (HIV), hepatitis C virus (anti-HCV), and syphilis (RPR). Nevertheless, this product should be handled using the same safety precautions used when handling potentially infectious material.
RECONSTITUTION & USE:
TO ENSURE PROPER SOLUBILITY OF HFN THIS RECONSTITUTION PROCEDURE MUST BE FOLLOWED.
Equilibrate vial to room temperature. Resuspend in sterile distilled water to a concentration of 1mg/ml. Allow 30 minutes for material to go into solution. DO NOT AGITATE OR SWIRL. If entire amount of material is not to be used immediately, transfer into appropriate aliquots and store at −20°C. It is recommended that solubilized product is used within two weeks. DO NOT STORE IN FROST-FREE FREEZER. AVOID MULTIPLE FREEZE THAWS.
Human Fibronectin is generally used in the concentration range of 1-5 micrograms per cm² of growth surface for attachment or at 5 micrograms per ml as a media additive.
Coating Procedure
Use these recommendations as guidelines to determine the optimal coating conditions for your culture system.
- Dilute fibronectin to desired concentration using serum-free culture Ca²⁺, Mg²⁺ free medium or buffer at pH 7-9. The final solution should be sufficiently dilute so that the volume added will cover the surface evenly. Example: If the final coating concentration will be 5 µg/cm², dilute the material to 50 µg/ml and add 1 ml/35 mm dish, 3 ml/60 mm dish, etc. NOTE: Because of the CAPS component in the HFN preparation, buffers of media containing Ca²⁺ and/or Mg²⁺ added to the HFN may result in the formation of insoluble metal hydroxides. This will not occur if the buffering capacity of the diluent brings the pH to 8.0 or lower.
- Add appropriate amount of diluted fibronectin to culture surface.
- Incubate at room temperature for 1 hour.
- Aspirate remaining material.
- Rinse plates carefully with dH₂O - avoid scraping bottom surface.
- Plates are ready for use. They may also be stored at 2-8°C damp or air dried if sterility is maintained.
