|Matt Lewis||Department of Pathology
University of Liverpool
Collagen gel containing 3T3 fibroblasts (dermal equivalent for raft culture)
Ingredients for 6 x collagen matrices in a 6-well plate;
Pre-chill pipettes, keep collagen on ice
The collagen solidifies above 8ºC
Mix 1.5mL of 10x DMEM with 1.5mL of 10x reconstitution buffer,
keep on ice.
The J2-3T3s seem to survive this somehow
Using chilled pipette, add the 12mL of collagen gently to the
cells and tilt to mix, avoiding bubbles as far as possible.
Everything is kept cold to avoid the collagen solidifying
Add 10N NaOH to bring the pH up to 7
Judge the pH visually by the phenol red in the DMEM
Pipette 22.5mL into each well and incubate O/N
Dissolve DMEM powder into 0.1 volume of H2O.
10x reconstitution buffer
2.2g sodium bicarbonate and 4.8g HEPES in 100mL H2O.
Collaborative Biomedical Products (part of Becton Dickinson) Cat # 354236