All about Collagen gels

Note: Collagen is derived from living rats and calf, whose connective tissue can vary and therefore the collagen can vary massively from batch to batch. If a series of experiments with collagen is planned, it is reasonable to use the same batch all the time (have a large enough stock!).

Normal Collagen gels(e.g. for invasion assays or 3D Migration)

Stock solution of concentration 2.4 mg/ml, KEEP EVERYTHING ON ICE

Amount is enough for 2 cell culture dishes (ø35mm)

1.2 ml Coll R (Collagen R, Serva, Heidelberg, Germany)

1.2 ml Coll G (Collagen G, Biochrom, Berlin, Germany)

270 µl 10x DMEM (Biochrom)

270 µl NaHCO3 (23 mg/ml stock solution)

43 µl NaOH

Mix everything together, be careful, avoid bubbles.

Put 1.2 ml stock solution per culture dish

Place it in the cell culture incubator (37°C, 5% CO2, 95 % humidity) !! Not on top of each other but side by side!!

Let it polymerize for 2hrs

After 2 hrs put 2ml PBS on it 

 

 

 

 

Solution to dilute collagen gels:

 

1/10  10xDMEM

 

1/10  NaHCO3

 

8/10 H2O (Millipore and sterile)

 

→ add NaOH until a pH of 10 is reached (test with pH sticks)

 

Stiffening of collagen gels:

 

For collagen gels prepared after protocol

 

Use 0.25 % glutaraldehyde in PBS

 

→ Put 2ml on top of the gel for 45 minutes

 

→ Wash several times (at least 24h) with 2 ml TRIS Buffer (20 mM)

 

→ before seeding cells, wash 2 times with DMEM

 

 

 

 

Fixiation of Collagen gels (e.g. to stop invasion assays)

 

For collagen gels prepared after protocol

 

Use 2.5 % glutaraldehyde in PBS.

 

→ Remove media

 

→ Put 2ml GA-solution on top of a collagen gel for 20 minutes

 

→ Remove the glutaraldehyde and put 2ml PBS on top of the gel

 

Note: Fixed collagen gels can be stored for a long time in the fridge. From time to time, make sure that there is still PBS on the gels to prevent dehydration.