Difference between revisions of "Cardiomyocyte Preparation"
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# Wash ventricles in 70% Leibovitz-15 (L15) medium (AL15) w/ pen-strep | # Wash ventricles in 70% Leibovitz-15 (L15) medium (AL15) w/ pen-strep | ||
# Store ventricles O.N. at 25C in AL15 w/ pen-strep | # Store ventricles O.N. at 25C in AL15 w/ pen-strep | ||
| − | # Digest ventricles 2ml d.sol | + | # Digest ventricles 2ml d.sol |
| − | |||
* aPBS w/ | * aPBS w/ | ||
* 0.5% Bactotrypsin (Difco) | * 0.5% Bactotrypsin (Difco) | ||
| Line 15: | Line 14: | ||
* 0.3% glucose | * 0.3% glucose | ||
* gentamycin (50ug/ml) | * gentamycin (50ug/ml) | ||
| + | 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) | # 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…) | # Pass through a 100um microsieve (reason for this???? And what to collect – flowthrough or the retenate… cardiomyocytes are min 150um in length…) | ||
Revision as of 08:44, 11 March 2015
(Bettencourt-Dias & Laube F combined)
Procedure:
- 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
- aPBS w/
- 0.5% Bactotrypsin (Difco)
- 380U/ml collagenase (Sigma)
- 0.15% BSA
- 0.3% glucose
- gentamycin (50ug/ml)
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.