Collagenase type II digestion- Full IMDM medium: Iscove’s modified Dulbecco’s medium
(IMDM), 10% (v/v) FBS, 2% (v/v) penicillin/streptomycin.
Warm full medium at 37C before use. - Collagenase type II solution: 0.5 g/L collagenase type II in
IMDM medium (seeNote 2). - Stirring water bath.
- 0.2μm filter.
Cardiomyocyte purification by Percoll gradient - 36% (v/v) Percoll (GE Healthcare) in IMDM medium: Mix
11.25 ml of Percoll gradient solution and 1 ml of sterile NaCl
(1.5 M). Use 7.8 ml of this mix and add 12.2 ml of warm
IMDM medium. Mix slowly and divide the solution into two
fractions of 10 ml in 50 ml tube.
Red blood cell lysis - Red blood cell (RBC) lysis buffer: 160 mM NH 4 Cl, 1 mM
EDTA, 10 mM KHCO 3 in sterile water. Filter the solution (see
Note 3). - 0.2μm filter.
Dish coating - Gelatin: 0.2% gelatin in sterile water (pre-warmed at 37C).
- Cell culture dishes: 3 cm diameter (9.6 cm^2 ).
Cell counting - Burker chamber.
- Trypan blue.
Cell plating - CO 2 incubator: Set to 5% CO 2 and 37C.
2.2 Evaluation
of Protein Synthesis
in Cardiomyocytes
During Hypertrophic
Treatment
Cardiomyocyte treatments- Cultured neonatal cardiomyocytes (750,000 cells per well of
9.6 cm^2 ).
2.^14 C-labeled L-phenylalanine (487 mCi/mmol, PerkinElmer). - Phenylephrine (PE) stock solution: 20 mM phenylephrine in
H 2 O. - Phenformin stock solution: 20 mM phenformin in H 2 O.
- AICAr stock solution: 20 mM AICAr in H 2 O.
- Full IMDM medium (seeSubheading2.1,item 6).
- IMDM medium without serum: IMDM, 2% (v/v) penicillin/
streptomycin (pre-warmed at 37C).
Cell harvest
324 Florence Mailleux et al.