Cell Attachment Factors

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Information

Growth and differentiation of anchorage dependent cells are strongly influenced by the glass or plastic culture flasks offered as substrate. Cell growth rates can often be improved by surface coating with attachment factors such as fibronectin, collagen, gelatine or polylysine. With a collagen coating, survival time of hepatocytes can be extended from normally one week to four weeks.
As another positive effect, proper coating may prevent from de-differentiation, a common phenomenon seen in many cell lines.

Collagen

For gel coating of flasks; from calf skin collagen, for in vitro use only
Successful geling is mainly influenced by the pH value in charge. Volumes given in the protocol may fluctuate from lot to lot to a certain extent; to determine the parameters of geling, medium RPMI 1640 (10x; cat.no. F 1223/F 1225) was used. Sterile technique is expected, and not described here in more detail.

Adjust all reagents to a temperature of +2 - +8°C prior to work.

- 1 M sodium hydroxide and 1 M HEPES buffer (cat.no. L 1613) are mixed equally (e.g. 5 ml sodium hydroxide with 5 ml HEPES buffer). This is referred to a Solution A.
- A 10x medium, and Solution A are mixed equally (e.g. 5 ml medium with 5 ml Solution A). This is referred to as Solution B.
- pH value should be 9.8 to 10.2; it is recommended to validate the method in an aliquot of the gel which was earlier removed.
- For the ready-to-use solution 8.0 ml of Collagen G are mixed carefully with 2.0 ml of Solution A; avoid air bubbles in the gel.
- 3.0 ml of the solution are applied for a 25 cm2 culture flask; for a 9 cm diameter petri dish, 5.0 ml are used. The culture flask is incubated for 24 hours at 35°C +/- 2°C; the gel should then be clear and rigid.

Pack/Density

Unit

Cat.no.


Collagen A 1 mg/ml Acid-soluble collagen from bovine placenta Storage: 24 months at +2 - +8°C	- / -	1x (6x5 ml)	L 7220



Collagen G For gel coating of flasks from calf skin collagen Storage: 24 months at +2 - +8°C	- / -	100 ml	L 7213

Fibronectin

Fibronectin from Human Plasma
Obtained from human citrate plasma, tested for the absence of HIV, and HBsAg. One unit contains 1 mg of deep-frozen fibronectin. Volume may slightly vary from lot to lot, and is indicated on each bottle.

Fibronectin from Bovine Plasma
Sterile and Purified by means of affinity chromatography. Each unit contains 1 mg dissolved, and deep-frozen bovine fibronectin. Volume may slightly vary from lot to lot, and is indicated on each bottle.

APPLICATION A:

- Thaw fibronectin in a +37°C water bath; dilute with sterile bi-distilled water (cat.no. L 0015) to a concentration of 0.02 mg/ml.
- Cover the bottom of a culture flask with the solution, and air-dry. Coated plates can be stored for a few weeks at room temperature.

APPLICATION B:
- Thaw fibronectin in a +37°C water bath; dilute with sterile PBS (cat.no. L 1813) to a concentration of 0.15 mg/ml.
- Add 1 ml of solution per 10 cm2 of culture flask; incubate at room temperature for 30 min.
- Remove solution and wash 1x with PBS; use culture flask immediately.

Pack/Density

Unit

Cat.no.


Fibronectin from bovine serum Storage: 24 months at -20°C	- / -	1 mg	L 7127



Fibronectin from human serum Storage: 24 months at -20°C	- / -	1 mg	L 7117

Gelatine

Porcine skin gelatine (approx. 300 Bloom) in distilled water.
- Liquify the gel in a +37°C water bath.
- Add 1 ml of solution per 10 cm2 of culture flask; incubate at +37°C for 30 min.
- Remove solution and wash 1x with sterile PBS; use culture flasks immediately.