Collagen Coating Protocol

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 (see Table 1).

Product No. Description Coating concentration Dilute in
11179179001 Collagen from rat tail tendon 5 μg/cm2 Sterile 0.2% acetic acid 
08-110 ECL Cell Attachment Matrix Sterile basal medium (without serum)
C0543 Collagen from Engelbreth-Holm-Swarm murine sarcoma basement membrane 6‑10 μg/cm2 0.25% Acetic acid
C2124 Collagen solution from bovine skin Sterile PBS
C2249 Collagen Solution from human fibroblasts Sterile PBS
C3867 Collagen, Type I solution from rat tail Sterile water
C4243 Collagen solution from bovine skin Sterile PBS
C5533 Collagen from human placenta 0.25% Acetic acid
C6745 Collagen Type IV from human cell culture Sterile HBSS
C7661 Collagen from rat tail 0.1 M acetic acid
C8919 Collagen from calf skin Sterile water
C9301 Collagen from chicken sternal cartilage 0.25% Acetic acid
C9791 Collagen from calf skin 0.1 M acetic acid
234184-M Collagen, Type II, Bovine Joint Cartilage 0.2 - 2 µg/cm2 500 mM acetic acid
08-115 Collagen Type I, rat tail 10 µg/cm2 Sterile PBS
122-20 Rat Tail Collagen Coating Solution (20 ML)   Ready-to-use
ECM104 Millicoat Human Collagen Type I Coated Strips (96-Wells)   Ready-to-use
ECM105 Millicoat Human Collagen Type IV Coated Strips (96-Wells)   Ready-to-use

Table 1: Collagens for cell attachment

Collagen Gels

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

Product No. Description
11179179001 Collagen from rat tail tendon
234138 Collagen, Type I, Human Skin
234154 Collagen, Type IV, Human Placenta
CC050 Human Collagen Type I
CC052 Human Collagen Type II
CC054 Human Collagen Type III
CC076 Human Collagen Type IV
CC077 Human Collagen Type V
CC078 Collagen Type III, bovine purified protein
CC083 Bovine Collagen Type IV
CC086 Bovine Collagen Type VI
5006 PureCol™
5074 PureCol™ EZ Gel solution
5168 Bovine Collagen
234149 Collagen, Type I, Human Placenta

Table 2: Collagens for gel preparation

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Frequently Asked Questions (FAQs)

  1. 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.

  2. Why am I seeing some insoluble particles in my collagen solution?
    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.

  3. What is the best solvent to dilute collagen?
    The solvents that are compatible to dilute collagens are provided in Table 1

  4. What is the optimum concentration of collagen for coating?
    The coating concentrations of collagens are suggested in Table 1. However, optimizing the concentrations that work for your cells is recommended.

  5. The product gelled while refrigerated. Can it still be used?
    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.

  6. Can all collagen products be used to make 3-D collagen gels?
    The collagen products that can be used to make gels are listed in Table 2.

  7. How long can collagen-coated plates be stored?
    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.

For additional questions contact our technical services here.