(Bettencourt-Dias & Laube F combined)
- Collect hearts in 70% Leibovitz-15 (L15) medium (AL15) w/ pen-strep
- Wash them once in 70% Leibovitz-15 (L15) medium (AL15) w/ pen-strep
- Remove the newt ventricles
- Wash ventricles in 70% Leibovitz-15 (L15) medium (AL15) w/ pen-strep
- Store ventricles O.N. at 25C in AL15 w/ pen-strep
- Digest ventricles 2ml d.sol (down under) for 8h in a shaking water bath at 27C. Change the enzyme solution every 1-2h.
- Neutralize cell suspensions w/ complete 4ml AL-15 medium (AL-15 w/ 10% FBS and pen-strep)
- Pass through a 100um microsieve (reason for this???? And what to collect – flowthrough or the retenate… cardiomyocytes are min 150um in length…)
- Centrifuge the neutralized suspensions for 10min at 500 rpm .
- Resuspend in complete AL15 (volume???)
- preplate in uncoated 6cm culture dishes for 3 days at 25C in a humidified CO2 incubator. Note: Blood cells, connective tissue and some pigmented cells attach to the culture dish during this step, while the cardiomyocytes remain in suspension (non-adhere)
- Transfer the supernatant onto laminin coated dishes (15ug/ml in serum-free medium, coating for 60 min) *at a density of 4000 cells/cm2* (is it necessary at such density?)
- Change the medium only when 3 days have passed since plating.
- aPBS w/
- 0.5% Bactotrypsin (Difco)
- 380U/ml collagenase (Sigma)
- 0.15% BSA
- 0.3% glucose
- gentamycin (50ug/ml)