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Collagen Attachment Protocols, Solubility, and Stability

Important points to remember before start

  1. Ensure the cells are healthy and in adequate numbers.
  2. Collagen solutions may be sterilized by dialysis in a 0.25% acetic acid and 0.5% chloroform solution.
  3. We do not recommend sterilizing the collagen solution by membrane filtration. We have found substantial protein loss by this method.
  4. Coating of tissue culture plastic dishes may be performed by air-drying. Dried coated dishes can be sterilized by exposure to UV light in a sterile culture hood or by rinsing with 70% ethanol.
  5. Surface coverage concentration may differ with the cells being cultured. Optimal conditions for attachment must be determined for each cell line and application. Recommended concentrations are listed in Table 1.
  6. Do not over-dry culture plates/coverslips post coating as this affects the attachment of cells.
  7. All the steps in the protocols need to be performed in sterile conditions.

Collagen Type I coating protocol for culture ware

  1. Add collagen to acetic acid (refer to Table 1) to obtain 0.1% (w/v) collagen solution. Stir at room temperature for 1-3 hours until dissolved. Collagen solution should be diluted 10-fold to obtain a working concentration of 0.01%.
  2. We recommend transferring the collagen solution to a glass bottle with a screw cap and carefully layering chloroform at the bottom. The amount of chloroform to use should be approximately 10% of the volume of collagen solution. DO NOT SHAKE OR STIR. Let stand overnight in the cold. Aseptically remove the top layer containing your collagen solution.
  3. Coat dishes with recommended concentrations in Table 1. Allow the protein to bind for several hours at room temperature, 37 °C, or overnight at 2-8 °C.
  4. Remove excess fluid from the coated surface and allow it to dry overnight.
  5. If the collagen solution is not sterile, the dried, coated surface can be sterilized by overnight exposure to UV light in a sterile tissue culture hood.
  6. Rinse with sterile tissue culture grade water or a balanced salt solution before introducing cells and medium.

Collagen Type II and IV coating protocol for culture ware

  1. Collagen Types II and IV may be reconstituted to a concentration of 0.5-2.0 mg/mL in acetic acid (refer to Table 1). Dissolve for several hours at 2-8 °C, occasionally swirling.
  2. Coat the tissue culture plastic dishes by air-drying. Alternatively, preincubate the coated culture ware overnight at 2-8 °C (or several hours at 37 °C).
  3. Dried coated dishes can be sterilized by exposure to UV light in a sterile culture hood or by rinsing with 70% ethanol.

Alternatively, we offer ready-to-use, collagen-coated 96-well strips (Table 1).

Table 1.Collagens for Cell Attachment

Collagen Gels

Products that are compatible for the preparation of collagen gels are in listed Table 2.

Table 2.Collagens for Gel Preparation

Frequently Asked Questions (FAQs)

Why does the vial of collagen solution look milky or contain particulates?

Due to cross-linkage and sequence variability, as well as structural changes that can occur during isolation and purification, Collagen Type I (C8919) is a heterogeneous mixture rather than a pure compound. This variability, along with slight differences in the raw material, lends itself to shifts in the opacity of the solution from lot to lot. Products listed as sterile is tested for sterility prior to release for sale to our customers. No further filtration of such products is necessary. A milky or particulate appearance is not indicative of microbial contamination and does not affect product use.

The solubility specification for some collagen products, like C7661, is clear to hazy colorless solution with a few insolubles at 1 mg/mL in water with 2 μL acetic acid (or 0.1 N acetic acid). The insolubles can be removed by settling or centrifugation.

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

What is the optimum concentration of collagen for coating?

This can occur but does not harm the product. When warmed to room temperature, the product will liquify. Please make sure it is well mixed prior to dilution for coating culture ware.

The collagen products that can be used to make gels are listed in Table 2.

We make no recommendations about the storage of plates after coating. Because several factors, such as humidity, may impact on the longevity of coated plates, customers should make their own determination as to whether plates can be stored and under which conditions. The product should not be exposed to temperatures above 50 °C.

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