CELL ATTACHMENT FACTORS

| Fibronectin | Collagen A | Collagen G | Gelatine | Polylysine |

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Growth and differentiation of anchorage dependant 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.

FIBRONECTIN FROM HUMAN PLASMA

Storage: -20⁰C

Obtained from human citrate plasma, tested for the absence of HIV, and HB_sAg. 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

Storage: -20°C

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.

	Unit	Cat. No.
Fibronectin from human plasma	L7117	1 mg/bottle
Fibronectin from bovine plasma	L7127	1 mg/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 fibronecin in a +37^oC wather bath; dilute with sterile PBS (cat. no. L 1813/L1815) 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,

COLLAGEN A

Storage: +2-+8°C

Acid-soluble collagen from bovine placenta

· Add an equal volume or sterile PBS (cat. no, L 1813 / L 1815) to the collagen.

· Add 1 ml of per 10 cm² or culture flask and Incubate at +35-+37°C for 30 min

· Remove solution and wash ix with PBS; use culture flasks immediately

	Cat. No	Unit
Collagen A (1mg/ml)	L7220	1 x (6x5 ml)

COLLAGEN G

Storage: +2 - +8^oC

For gel coating of flasks; from calf skin collagen, for in vitro use only

Reference:

Riemschneider, R. et al., Pharm. Ind. 41, 1077 [1979]: 44, 745 [1982]

Description	Cat. No
HEPES	L1613
Water	L0015
(10x) medium, e.g. RPM1 1640	F1225
Collagen G	L7213
NaOH, pH-paper

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. F1223 /F1225) was used. Sterile technique is expected.

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

· 1 M sodium hydroxide and 1 M HEPES buffer (cat.no. L1613j are mixed equally (e.g. 5 ml sodium hydroxide with 5 ml HEPES buffer). This is referred to as 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.

	Cat. No	Unit
Collagen G	L7213	100 ml

GELATINE

Storage: +2-+8°C

Porcine skin gelatine (approx. 300 Bloom) in distilled water.

· Liquify the gel in a +37^oC

· Add 1 ml of solution per 10 cm² of culture flask; Incubate at +37°C for 30 min.

· Remove solution and wash Ix with sterile PBS; use culture flasks immediately.

	Cat. No	Unit
Gelatine (l0mg/ml)	L7230	l x (6 x 5ml)

POLYLYSINE

Storage: +2 - +8°C

The preparation contains 0.1 mg/ml poly-L-lysine as hydrobromide ( MW > 300 000).

· Add 1 ml of per 10 cm2 of culture flask; incubate at room temperature for 30 min.

· Remove the solution and wash 2x with PBS (cat. no. L1813 /L1815).

	Cat. No.	Unit
Polylysine	L7240	1 x (6x5 ml)