Cardiomyocyte Preparation: Difference between revisions

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(Created page with "Bettencourt-Dias & Laube F combined 1) Collect hearts in 70% Leibovitz-15 (L15) medium (AL15) w/ pen-strep 2) Wash them once in 70% Leibovitz-15 (L15) medium (AL15) w/ pen-st...")
 
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Bettencourt-Dias & Laube F combined
(Bettencourt-Dias & Laube F combined)


1) Collect hearts in 70% Leibovitz-15 (L15) medium (AL15) w/ pen-strep
== Procedure ==
2) Wash them once in 70% Leibovitz-15 (L15) medium (AL15) w/ pen-strep
# Collect hearts in 70% Leibovitz-15 (L15) medium (AL15) w/ pen-strep
3) Remove the newt ventricles
# Wash them once in 70% Leibovitz-15 (L15) medium (AL15) w/ pen-strep
4) Wash ventricles in 70% Leibovitz-15 (L15) medium (AL15) w/ pen-strep
# Remove the newt ventricles
5) Store ventricles O.N. at 25C in AL15 w/ pen-strep
# Wash ventricles in 70% Leibovitz-15 (L15) medium (AL15) w/ pen-strep
6) Digest ventricles 2ml d.sol for 8h in a shaking water bath at 27C. Change the enzyme solution every 1-2h.
# Store ventricles O.N. at 25C in AL15 w/ pen-strep
Note: D.sol
# Digest ventricles 2ml d.sol (down under) for 8h in a shaking water bath at 27C. Change the enzyme solution every 1-2h.  
- aPBS w/
# Neutralize cell suspensions w/ complete  4ml AL-15 medium (AL-15 w/ 10% FBS and pen-strep)
- 0.5% Bactotrypsin (Difco)
# Pass through a 100um microsieve (reason for this???? And what to collect – flowthrough or the retenate… cardiomyocytes are min 150um in length…)
- 380U/ml collagenase (Sigma)
# Centrifuge the neutralized suspensions for 10min at 500 rpm .
- 0.15% BSA
# Resuspend in complete AL15 (volume???)
- 0.3% glucose
# 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)''
- gentamycin (50ug/ml)
# 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.


7) Neutralize cell suspensions w/ complete  4ml AL-15 medium (AL-15 w/ 10% FBS and pen-strep)
 
8) Pass through a 100um microsieve (reason for this???? And what to collect – flowthrough or the retenate… cardiomyocytes are min 150um in length…)
D.sol:
9) Centrifuge the neutralized suspensions for 10min at 500 rpm .
* aPBS w/
10) Resuspend in complete AL15 (volume???)
* 0.5% Bactotrypsin (Difco)
11) preplate in uncoated 6cm culture dishes for 3 days at 25C in a humidified CO2 incubator. 
* 380U/ml collagenase (Sigma)
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)
* 0.15% BSA
12) 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?)
* 0.3% glucose
13) Change the medium only when 3 days have passed since plating.
* gentamycin (50ug/ml)

Latest revision as of 09:19, 25 March 2015

(Bettencourt-Dias & Laube F combined)

Procedure

  1. Collect hearts in 70% Leibovitz-15 (L15) medium (AL15) w/ pen-strep
  2. Wash them once in 70% Leibovitz-15 (L15) medium (AL15) w/ pen-strep
  3. Remove the newt ventricles
  4. Wash ventricles in 70% Leibovitz-15 (L15) medium (AL15) w/ pen-strep
  5. Store ventricles O.N. at 25C in AL15 w/ pen-strep
  6. Digest ventricles 2ml d.sol (down under) for 8h in a shaking water bath at 27C. Change the enzyme solution every 1-2h.
  7. Neutralize cell suspensions w/ complete 4ml AL-15 medium (AL-15 w/ 10% FBS and pen-strep)
  8. Pass through a 100um microsieve (reason for this???? And what to collect – flowthrough or the retenate… cardiomyocytes are min 150um in length…)
  9. Centrifuge the neutralized suspensions for 10min at 500 rpm .
  10. Resuspend in complete AL15 (volume???)
  11. 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)
  12. 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?)
  13. Change the medium only when 3 days have passed since plating.


D.sol:

  • aPBS w/
  • 0.5% Bactotrypsin (Difco)
  • 380U/ml collagenase (Sigma)
  • 0.15% BSA
  • 0.3% glucose
  • gentamycin (50ug/ml)
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