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General description
This product is identical to Biochrom GmbH part number L 7213. It is comprised of acid-soluble calfskin collagens in a 0.4 % solution containing 15 mmol/l HCl. Collagen G is used in the production of gels, to embed cells. Collagen G gels are suitable for use as a substrate for adherent cells in a cell culture vessel and as a floating matrix in or on cell culture medium.
Preparation Note
Recommendations for optimum gelling with Collagen G
Geling is heavily influenced by the pH value. Before starting work, pre-cool all reagents to a temperature of +2 – +8C.
Solution A:
0.7 M sodium hydroxide and 1 M HEPES buffer are mixed equally (e.g. 5 ml sodium hydroxide with 5 ml HEPES buffer).
Solution B:
A 10x medium (e.g. RPMI 1640) and solution A are mixed equally (e.g. 5 ml medium with 5 ml solution A).
The pH of solution B should be between 7.90 and 8.05. It is advisable to check the pH using a suitable measuring method. If the gel should remain sterile, measure the pH of an aliquot previously removed.
8.0 ml Collagen G is gently mixed with 2.0 ml of solution B to produce the ready-to-use solution for gelling. This prevents the formation of air bubbles.
3.0 ml of solution prepared in this way is pipetted into a 25 cm2 culture flask. For a culture flask with a diameter of 9 cm, 5.0 ml is required. The culture flask is then incubated vibration-free for at least 1 hour or, for optimum stability, 24 hours at +35°C (± 2°C).
Examining the gel produced under a microscope and taking pictures
If a 35 mm culture dish is filled with 2 ml Collagen G/buffer media mixture (pH approx. 7.2 – 7.8) and incubated, a gel with faint white diffusion is formed, viewed from above. This diffusion appears as a result of crosslinking of the collagen molecules and should not cause any problems when examining under a microscope or taking pictures.
The accumulation of diffusion is proportional to the increase of thickness of the gel, for example if 5-7 ml instead of 2 ml per 10 cm2 Collagen G (35 mm culture vessel) is used. This could make microscopic and photographic analysis difficult.
Other Notes
Specifications:
Cell Attachment: Pass
pH: 1.8-2.4
Osmolality: 0.01-0.05 Osm/kg H20
Collagen Content: 0.37-0.45%
Hydroxyproline: 0.046-0.0565%
Color: Colorless
Appearance: Clear
