Collagen gel containing 3T3 fibroblasts (dermal equivalent for raft culture) Ingredients for 6 x collagen matrices in a 6-well plate; Roughly 3x106 J2-3T3s (a fully confluent T75?) 1.5ml 10x reconstitution buffer 1.5ml 10x DMEM 12ml rat tail type 1 collagen (>3.8mg/ml) 10N NaOH Glacial acetic acid (in case) Method Pre-chill pipettes, keep collagen on ice The collagen solidifies above 8C Mix 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 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 Avoid bubbles Add 10N NaOH to bring the pH up to 7 Judge the pH visually by the phenol red in the DMEM Maybe 30-60ml will be necessary Don’t go too far, use the glacial AcCOOH if you have to, but the more mixing the more bubbles. Pipette 2 – 2.5ml into each well and incubate O/N In the morning, add 2ml raft media on top of each matrix. Use within 1 week, change media every 2 days. Buffers 10x DMEM Dissolve DMEM powder into 0.1 volume of H2O. Filter sterilise and store at –20C in working aliquots. (It looks yellow and doesn’t dissolve completely). 10x reconstitution buffer Dissolve 2.2g sodium bicarbonate and 4.8g HEPES in 100ml H2O. Filter sterilise and store at –20C in working aliquots. Suppliers Collagen Collaborative Biomedical Products (part of Becton Dickinson) Cat # 354236 Telephone (UK) 01865 781615 It comes in vials of 100mg dissolved in 0.02N HCl at roughly 4mg/ml (ie 25ml). Try to get a batch of at least 3.8mg/ml. If it is stronger than 4mg/ml then dilute it to 4mg/ml with 0.02N acetic acid.