Author: Matt Lewis
Ingredients for 6 x collagen matrices in a 6-well plate
- 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)
- 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.
- 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.