|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 8CMix 1.5ml of 10x DMEM with 1.5ml of 10x reconstitution buffer, keep on ice.
Count J2-3T3s and pellet required number in a universal.
Add the 3ml of [1:1, 10x DMEM and 10x reconstitution buffer] and swirl to resuspend the cells, keep on ice
The J2-3T3s seem to survive this somehowUsing 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
Judge the pH visually by the phenol red in the DMEM
In the morning, add 2ml raft media on top of each matrix.
Use within 1 week, change media every 2 days.
Dissolve DMEM powder into 0.1 volume of H2O.
10x reconstitution buffer
Dissolve 2.2g sodium bicarbonate and 4.8g HEPES in 100ml H2O.
Collaborative Biomedical Products (part of Becton Dickinson) Cat # 354236