Acid-soluble collagen from bovine placenta

• Add an equal volume of sterile PBS to the collagen.
• Add 1 ml per 10 cm2 of culture flask and incubate at +35 - +37° C for 30 min.
• Remove solution and wash 1x with PBS; use culture flasks immediately.

In monolayer culture, normal human and murine liver cells were successfully grown for a period of up to one week, provided that the culture flasks were coated with collagen A. Porcine cells, however, do not require such coating with the same settings.
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.

Storage: +2 - +8° C

0,2 % sterile solution:
Type 1 rat tail collagen; 2 mg/ml in 0,1 % acetic acid. Excellent substrate for the culture of hepatocytes, fibroblasts and epithelial cells.

0,4 % sterile solution:
Type 1 rat tail collagen; 4 mg/ml in 0,1 % acetic acid. Excellent substrate for the culture of hepatocytes, fibroblasts and epithelial cells.

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

Components

Hepes
Water
10x-medium, e. g. RPMI 1640
Collagen G
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. F 1223/F 1225) was used. Sterile technique is required, 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 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.

Storage: +2° C - +8° C

This highly purified preparation of mouse Laminin I increa-ses cell adhesion, migration, growth, and differentiation. It is composed of α1β1γ1 chains with a total MW of 800 kD and is used for the coating of culture dishes.

Source: Murine Engelbreth-Holm-Swarm (EHS) tumor.
Storage Buffer: Dulbecco‘s Modified Eagle‘s Medium with 10 μg/ml gentamycin sulfate.
Storage: Store at 20° C or at 80° C in a manual defrost freezer.
Purity: Purity > 90 % by SDS-PAGE.

Specifications:

Functional Assays:

• Supports the formation of neuronal filaments of NG108-15 cells in a neurite outgrowth assay.

Sterility Testing:

• No bacterial or fungal growth detected after incubation at 37° C for 14 days following USP XXIV, Chapter 71 sterility testing.
• No mycoplasma contamination detected by PCR.
• Endotoxin concentrations < 20 EU/ml by LAL assay.

Coating Procedure:
The recommended working concentration is 0.05 - 10 μg/ cm2 of growth surface (0.05 - 10 μg/ml) depending on cell type.

a. Thaw stock solution on ice for several hours. Place plates on ice and prechill pipette tips. In an laminar flow hood, dilute appropriately with cold tissue culture plates. Spread the solution to completely cover the bottom of the wells.

b. The following table is a guide for the suggested volumes required per well:

c. Incubate the plates at 37° C for 1 hour. In the laminar flow hood, remove excess liquid from the wells of the tissue culture plate.

Rinse the wells once with tissue culture medium and then add your cells.

The gelatine solution is used for coating cell culture dishes. It is applied in cell culture at work with for instant endothelial cells or ES-cells.