REAGENTS AND SUPPLEMENTS

Normal attachment, growth, and development of many cell types are dependent on attachment factors and extracellular matrix components. While some cells are able to synthesize these components, others require an exogenous source, particularly when grown in serum-free culture.

To help facilitate attachment, cell spreading, growth, morphology, differentiation, and motility of your cells, Sigma offers an extensive line of attachment and matrix factors. Each lot is cell culture tested to assess its ability to promote cell attachment and spreading.

Collagen
Chondroitin Sulfate A
ECM gel
Fibronectin
Gelatin
Laminin
Superfibronectin
Thrombospondin
Vitronectin
Proteoglycans
Poly-Lysine
Disintegrins
Fibronectin Proteolytic Fragments

Collagen Type I
Target cells for attachment:

• Epithelial cells
• Muscle cells
• Nerve cells

Product Use

Collagen Type I, Acid soluble powder

Collagen Type I, 0.1% Solution, Sterile-filtered
(Not suitable for 3D gel formation)

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Collagen Type II
Target cells for attachment:

• Chondrocytes

Product Use

Collagen Type II, Powder

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Collagen Type IV
Target cells for attachment:

• Epithelial cells
• Enodothelial cells
• Muscle cells
• Nerve cells

Product Use

Collagen Type IV, Powder

Collagen Type IV, Lyophilized

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Chondroitin Sulfate A
Appears to play a regulatory role for:

• Chondrocytes
• Neural cells
• Some tumor cells

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ECM gel
Target cells for attachment:

• Epithelial cells
• Enodothelial cells
• Muscle cells
• Nerve cells
• Tumor cells

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Fibronectin
Target cells for attachment:

• Epithelial cells
• Mesenchymal cells
• Neuronal cells
• Fibroblasts
• Neural crest cells
• Endothelial cells

Product Use

Fibronectin, 0.1% Solution

Fibronectin, Fragment III₁ -C

Fibronectin, Lyophilized

Fibronectin Cellular, Lyophilized

Fibronectin Cellular, Aseptically Processed, Lyophilized

Fibronectin-Like Engineered Protein Polymer

Fibronectin-Like Engineered Protein Polymer-plus
Genetically Engineered, Lyophilized, Autoclaved

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Gelatin
Used for attachment of a variety of cell types

Product Use

Gelatin 2% Solution, Approx. 225 Bloom, Autoclaved

Gelatin, Approx. 225 Bloom

Gelatin, Approx. 300 Bloom

Gelatin, Approx. 300 Bloom, Lyophilized, Sterilized by g-irradiation

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Laminin
Target cells for attachment:

• Epithelial cells
• Endothelial cells
• Muscle cells
• Tumor cells
• Hepatocytes
• Schwannoma

Product Use

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Superfibronectin
Target cells for attachment:

• Epithelial cells
• Mesenchymal cells
• Neuronal cells
• Fibroblasts
• Neural crest cells
• Endothelial cells

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Thrombospondin, Lyophilized
Target cells for attachment:

• Osteoblasts
• Bovine aortic endothelial cells
• Neurons
• Human melanoma cells

Product Use

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Vitronectin
Cells with integrin receptors that bind vitronectin:

• Platelets
• Endothelial cells
• Melanoma cells
• Osteosarcoma

Product Use

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PRODUCT USE

Collagen Type I (Product Nos. C 1809, C 7661, C 9791, C 8919*)
Optimal conditions for attachment must be determined for each cell line and application.

1. Add collagen to 0.1 M acetic acid to obtain 0.1% (w/v) collagen solution. Allow to stir at room temperature 1-3 hours until dissolved. Collagen solution (Product No. C 8919) should be diluted 1:10 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. Allow to sit overnight in the cold. Aseptically remove the top layer containing your collagen solution. We do not recommend sterilizing the collagen solution by membrane filtration. We have found substantial protein loss by this method.
3. Coat dishes with 6-10 µg/cm². 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. If the collagen solution is not sterile, the dried, coated surface can be sterilized easily by overnight exposure to UV light in a sterile tissue culture hood.
5. Rinse with sterile tissue culture grade water or a balanced salt solution before introducing cells and medium.

*Note: Steps 1 and 2 are not necessary for the Collagen Solution, Product No. C 8919.

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Collagen Type II and Type IV (Product Nos. C 9301, C 0543, C 5533)
Optimal conditions for attachment must be determined for each cell line and application.

1. Collagen Types II and IV may be reconstituted to a concentration of 0.5-2.0 mg/ml in 0.25% acetic acid. Dissolve for several hours at 2-8°C, occasionally swirling.
2. Coating of tissue culture plastic dishes may be performed by air drying the above protein solution, or by preincubating the same solution overnight at 2-8°C (or several hours at 37°C) without air drying.
3. Dried coated dishes can be sterilized overnight by exposure to UV light in a sterile tissue culture hood or by rinsing with 70% ethanol. Alternatively, the collagen solution may be sterilized by dialysis in a 0.25% acetic acid and 0.5% chloroform solution.

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Fibronectin (Product Nos. F 1141*, F 0895*, F 4759, F 2006, F 0635)
Optimal conditions for attachment must be determined for each cell line and application.

1. Reconstitute with 1 ml sterile H₂O/mg of protein. Allow to dissolve for at least 30 minutes at 37°C. A small amount of undissolved material may remain. This will not affect product performance.
2. Dilute fibronectin in sterile balanced salt solution and coat the culture surface with a minimal volume.
3. Allow to air dry for at least 45 minutes at room temperature. Excess fibronectin may be removed by aspiration, but is not necessary.
   *Note: Step 1 is not necessary for the Fibronectin Solutions, Product Nos. F 1141 and F 0895.
4. Freezing and thawing of reconstituted fibronectin is not recommended as breakdown of protein will occur.

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Cellular Fibronectin (Product Nos. F 2518, F 6277)
Optimal conditions for attachment must be determined for each cell line and application.

1. Reconstitute with 0.5 ml of sterile water. Do not agitate. Allow the solution to stand 30 minutes to solubilize. A small amount of undissolved material may remain. This will not affect product performance.
2. Dilute fibronectin in sterile balanced salt solution and coat the culture surface with a minimal volume.
3. Allow to air dry for at least 45 minutes at room temperature. Excess fibronectin may be removed by aspiration, but is not necessary.

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Fibronectin-Like Engineered Protein Polymer (Product No. F 5022)
Optimal conditions for attachment must be determined for each cell line and application.
The diluent and the stock solution contain 4.5 M LiClO₄ which is near saturation at room temperature. During shipping (diluent) and storage (diluent and stock solution) it is likely that there will be some recrystallization in the vials. Warming will return the crystals into solution. Store tightly sealed at room temperature.

1. Reconstitute the lyophilized product to a 1 mg/ml stock solution by combining with the provided diluent. Swirl occasionally until dissolved.
2. Dilute to working concentration with a balanced salt solution in the range of 1:10 to 1:50, depending upon the cell line and application. The optimal range may be established by titer dilution.
3. Working concentration range is 2-10 µg/cm². If currently using fibronectin, use the polymer at the same concentration. Some cell lines show equivalent performance using from 2 to 5 times less protein polymer than fibronectin.
4. Coat surfaces with 0.1 ml/cm² of the working solution for 5 minutes at room temperature.
5. Remove the coating solution and immediately rinse twice with a balanced salt solution or tissue culture medium. Coated substrates can be used immediately or stored for up to 4 months at room temperature.

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Gelatin Solution, 2% (Product No. G 1393)
Optimal conditions for attachment must be determined for each cell line and application.

1. Allow gelatin solution to completely liquefy at 37°C.
2. Coat culture surface with 5-10 µl gelatin solution/cm² (i.e., 0.1-0.2 mg/cm² gelatin).
3. Allow to dry at least 2 hours before introducing cells and medium.

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Gelatin (Product Nos. G 9391, G 1890, G 9136)
Optimal conditions for attachment must be determined for each cell line and application.

1. Prepare a 2% (w/v) solution by dissolving gelatin in tissue culture grade water.
2. Sterilize by autoclaving at 121°C, 15 psi for 30 minutes.
3. Coat culture surface with 5-10 µl gelatin solution/cm² (i.e. 0.1-0.2 mg/cm² gelatin).
4. Allow to dry at least 2 hours before introducing cells and medium.

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Laminin (Product Nos. L 2020, L 6274)
Optimal conditions for attachment must be determined for each cell line and application.

1. Slowly thaw laminin at 2-8°C to avoid the formation of a gel.
2. Dilute in a balanced salt solution and coat culture surface with a minimal volume.
3. Allow to air dry at least 45 minutes before introducing cells and medium.

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Thrombospondin (Product No. T 7043)
Optimal conditions must be determined for each cell line and application.

1. Reconstitute with 0.5 ml of tissue culture grade water and sterilize by filtration.
2. The resulting solution is slightly hazy and has a concentration of 40 µg/ml.
3. Use concentration for thrombospondin in culture has been reported in the range of 25 ng to 50 µg/ml depending upon the application.
4. Please refer to the literature for more specific information.

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Vitronectin (Product Nos. V 8379, V 0132, V 9881)
Optimal conditions must be determined for each cell line and application.

1. Reconstitute with tissue culture grade water and sterilize by filtration.
2. Material is reported to be active at a concentration of 0.1 mg/cm² of surface area. Optimal concentrations vary with each cell line.
3. Coat culture surface for 1-2 hours at 37°C. Remove any remaining solution and wash with a balanced salt solution before introducing cells and medium.

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