Fibronectin Coating Protocol

Important points to remember before start

  1. Ensure the cells are healthy and in adequate numbers.
  2. Fibronectin products provided as solutions can be stored for at least a year at 2 - 8  °C if kept sterile.
  3. Reconstitute fibronectin products provided as lyophilized powders in suitable solvents. Dissolve for at least 30 minutes at 37 °C. A small amount of undissolved material may remain. This will not affect product performance. Reconstituted fibronectin solution can be stored in working aliquots at -20 °C or lower.
  4. Vortexing or excessive agitation of fibronectin solutions should be avoided.
  5. Fibronectin coated cultureware can be stored for 2-4 weeks at 2-8 °C in a closed sterile container or sterile sealable bags.
  6. Surface coverage concentration may differ with the cells being cultured. Protocol optimization is recommended to determine actual coating concentration for your cells. Recommended concentrations are listed in Table 1.
  7. Do not over-dry culture plates/coverslips post coating as this affects the attachment of cells.
  8. All the steps in the protocols need to be performed in sterile conditions.

Fibronectin coating protocol for culture ware

  1. Dilute fibronectin to the desired concentration. Optimum conditions for attachment are dependent on cell type and application. The typical coating concentration is 1 – 5 ug/cm2.
  2. Coat culture surface with a minimal volume.
  3. Once the entire area is covered, remove excess solution. This can be used to coat another well/plate.
  4. Air dry at least for 45 minutes at room temperature. before introducing cells and medium.
Table 1.Fibronectins for cell attachment.

Fibronectin Proteolytic Fragments

Fibronectin fragments are obtained from plasma fibronectin by using proteolytic enzymes. These fragments are useful tools for mapping regions within the fibronectin molecule, which are responsible for specific binding to collagen and for other biologically relevant functions.

Table 2.Fibronectin fragments for binding studies.

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Frequently Asked Questions (FAQs)

What is the purity of fibronectin products?

We do not determine the purity of fibronectin solutions. An SDS PAGE gel is run and one major fibronectin band of 120kDa is compared to past lots to ensure consistent quality. All our fibronectin powders are ≥95% pure.

The solvents that are compatible to dilute fibronectins are provided in Table 1.

Fibronectin products provided as solutions can be stored for a year at 2 - 8 °C provided they are sterile. Diluted, unused fibronectin can be kept at 2 - 8 °C for up to 1 week. Fibronectin-coated plates can be stored at 2 - 8 °C for up to 4 weeks provided they are sealed well to prevent contamination and/or drying up of fibronectin. Do not use the product if discoloration or cracks appear on the surface of the coated material.

The coating concentrations of our fibronectins are suggested in Table 1. However, optimizing the concentrations that work for your cells is recommended.

While laminin and fibronectin are widely used ECM proteins, the one that your cell needs should be determined by test experiments. Stem cells express varied integrins on the surface during different stages of differentiation. The choice of ECM protein would depend on the stage of differentiation you are interested in studying.