- Add an aliquot of 8–10 ml collagenase type II solution on the
cardiomyocytes, and shake the flask in stirring bath at 37C for
1 min. - Shake the flask vigorously during 10 s. Under the hood,
remove the supernatant which is collected into a 15 ml tube.
Keep this tube on ice. - Add the second aliquot of 8–10 ml collagenase type II solution
on the remaining cardiomyocytes in the flask and shake in
stirring bath at 37C for 15 s. - Shake the flask vigorously during 10 s. Under the hood,
remove the supernatant which is collected into another 15 ml
tube. Keep this tube on ice. - Repeatsteps 5and 6 until hearts are fully digested, increasing
number of 15 ml tubes. - Adjust the volume of all tubes to the same volume for correct
centrifugation. - Centrifuge at 183gfor 10 min at 4C.
- Discard the supernatant of each tube (seeNote 7). Resuspend
all the pellets in a total of 10 ml of warm full IMDM medium,
and pool all resuspended pellets in a 50 ml tube. - Homogenize the solution with a 25 ml pipette.
Cardiomyocyte purification by Percoll gradient- Slowly, add half of the cardiomyocyte solution on 10 ml of
Percoll gradient solution (seeNote 8). - Add the other half of cardiomyocyte solution on top of Percoll
gradient solution. - Centrifuge at 1650gfor 30 min at 15C without braking.
- Remove the pellet containing cardiomyocyte and red blood
cells with a 5 ml pipette, and put it in a new 50 ml tube (see
Note 9).
Red blood cell lysis
- Add 5 ml of RBC lysis buffer on cardiomyocytes and agitate
while keeping away from light for 5 min at room temperature
in a stirring plate. - Add 45 ml of HBSS on the solution containing cardiomyocytes
and RBC lysis buffer. - Centrifuge for 5 min at 183gat 4C.
- Discard supernatant and add 50 ml of HBSS, invert the tube,
and centrifuge again for 5 min at 183gat 4C. - Resuspend the cardiomyocytes in 3.5 ml of warm full IMDM
medium.
Study of Cardiac Hypertrophy and Protein Synthesis 329