Using a digestive enzyme

  1. Remove media from around and on top of gel, wash 3 x 5 minute incubations with PBS.
  2. Remove gel from insert and add to homogeniser tube
  3. Add 400uL of pronase solution (10mg/mL stock) (Sigma)
  4. Triturate the gel
  5. Incubate in water bath at 37°C for 5 minutes, with agitation every minute
  6. Add buffer RLT from RNeasy mini kit (350μl for <5×106 cells)
  7. Homogenise with an automated homogeniser (our lab uses IKA RW16 basic, Sigma)

Diluting the synthetic ECM

  1. Remove media from around and on top of gel, wash 3 x 5 minute incubations with PBS.
  2. Remove gel from insert and add to a falcon tube
  3. Dilute a 1000 times the hydrogels with cell culture media or saline solution
  4. Centrifuge at 4500rpm for 5min
  5. Collect the cells platelets at the bottom of the falcon tube