COLLAGEN

Collagen may be used to coat glass coverslips for the growth of epithelial, endothelial and muscle cells, neurons, PC12 and CHO cell lines. Type I collagen is most often used for coating substrates for cell culture because it is easily obtainable from rat tails. For short term cultures, collagen can be simply applied to glass coverslips and allowed to dry.
1. Dilute collagen solution 1:10 - 1:50 with 30% ethanol and spread over surface of sterile glass coverslip.
2. Air dry in a tissue culture hood.
3. Cells can be seeded directly on the collagen surface.
4. Collagen coating prepared in this way tends to detach from the glass in long-term cultures.

   Collagen IV is the major constituent of basement membrane and is therefore a more physiological coating for the culture of many cell types. For long-term cultures, collagen I and IV can be applied to glass coverslips by first coating the glass with polylysine or polyornithine. This provides a more stable collagen coating.
1. Prepare polylysine or polyornithine (MW of 30,000 -70,000) at 0.1-1 mg/ml in 0.15 M borate buffer (pH 8.3).
Filter sterilize.
2. Add enough solution to pool over surface of sterile glass coverslip.
3. Incubate 2-24 hours at room temperature.
4. Aspirate solution and wash coverslips 3 times with water.
5. Pool collagen solution, 100 ug/ml in water over surface of coverslip.
6. Incubate 4 - 16 hours.
7. Rinse once with media and seed with cells. Alternatively, for long-term cultures, double layered collagen coatings can
provide a stable coating.

Alternatively, for long-term cultures, double layered collagen coatings can
provide a stable coating.

1. Spread a couple of drops of sterile collagen I solution on the sterile glass coverslip.
2. Immediately neutralize for 2 minutes with ammonium hydroxide vapors by placing the dish of coverslips in a
covered dish containing filter paper wet with concentrated ammonium hydroxide. This will cause the collagen to gel.
3. Wash coverslips twice with sterile water.
4. Gently spread a couple of drops of collagen over the surface of the gelled collagen and air dry.
5. Use within a few hours for cell culture.

Gelatin can also be used for the culture of some cell types including glial cells.
1. Dissolve 100 mg gelatin in 100 ml water (triple glass
distilled or RO).
2. Autoclave to sterilize.
3. While hot, thoroughly mix gelatin solution.
4. Add enough solution to pool over surface of sterile glass coverslip.

5. Chill for 2-24 hours at 4oC.
6. Remove gelatin by aspiration and add sterile water.
7. Dishes can be stored for up to one week at 4oC. Remove water immediately before use for cell culture

POLYLYSINE AND POLYORNITHINE
Nearly all types of cells adhere to these polymers of basic amino acids. They are particularly useful for the culture of CNS neurons. The L- or D-isomers can be used for cell attachment, however, the D-isomer may be preferred because it
is not subject to breakdown by proteases released by cells.
    Prepare polylysine or polyornithine (MW of 30,000 - 70,000) at 0.1-1 mg/ml in 0.15 M borate buffer (pH 8.3).
Filter sterilize.
    Add enough solution to pool over surface of sterile glass coverslip.
    Incubate 2-24 hours at room temperature.
    Aspirate solution and wash coverslips 3 times with media or PBS.
    Immediately add cell suspension or growth media.

FIBRONECTIN

     Fibronectin is an extracellular matrix constituent use for the culture of endothelial cells, fibroblasts, neurons and CHO cells.
    Stock solution can be prepared by dissolving 1 mg/ml fibronectin in PBS. Filter sterilize and freeze in aliquots.
    Diluted stock solution to 50-100 ug/ml in basal medium or PBS.
    Add enough solution to pool over surface of sterile glass coverslip.
    Incubate for 30-45 min at room temperature.
    Aspirate to remove fibronectin and rinse coverslips with media or PBS.
    Immediately add cell suspension or growth media. Do not allow coating to dry.

LAMININ
    Laminin is an extracellular matrix constituent used for the culture of neurons, epithelial cells, leukocytes, myoblasts and CHO cells.
1. Stock solution can be prepared by dissolving 1 mg/ml laminin in PBS. Filter sterilize and freeze in aliquots.
2. Diluted stock solution to 10-100 ug/ml in basal medium or PBS.
3. Add enough solution to pool over surface of sterile glass coverslip.
4. Incubate several hours at room temperature.
5. Aspirate to remove laminin and rinse coverslips with media or PBS.
6. Immediately add cell suspension or growth media. Do not allow coating to dry.
7. Coating the glass coverslip first with polylysine or polyornithine and then laminin may increase the concentration of laminin applied using this method.