Author: Matt Lewis ### Ingredients for 6 x collagen matrices in a 6-well plate 1. Roughly 3x10e6 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 1. 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. 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 –20ºC 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 –20ºC in working aliquots.