Dish coating- Add 1 ml of 0.2% gelatin solution in each dish and incubate for
15 min (seeNote 10). - Remove the solution and leave the dishes opened under the
hood until complete drying (seeNote 11).
Cell counting - Dilute 10μl of cardiomyocyte solution in 90μl of trypan blue
(dilution 1/10). - Put 10μl of this mix in the burker chamber.
- Count living (non-blue) cells (seeNote 12).
Cell plating - Dilute cell solution in an adequate volume of warm full IMDM
medium knowing that 750,000 cells per dish (9.6 cm^2 ) are
needed for protein synthesis and signaling analysis and wells
can contain 2 ml of medium. - Let cells to adhere overnight in incubator at 37C and 5%
CO 2.
3.2 Evaluation
of Protein Synthesis
in Cardiomyocytes
During Hypertrophic
Treatment
Cardiomyocyte treatments- The day after plating cardiomyocytes, the IMDM medium is
changed and replaced by 2 ml of pre-warmed full IMDM
medium for 24 h. - After 24 h, replace the medium by 2 ml of IMDM medium
without serum (pre-warmed at 37C) for 2 h before stimula-
tion (seeNote 13). - Treat the cells according to the following conditions: controls
(no treatment), 20μM PE (hypertrophic treatment), 1 mM
phenformin or 1 mM AICAr (AMPK stimulation), and
PEþphenformin or PEþAICAr (simultaneous hypertrophic
treatment and AMPK stimulation). At the same time, add
1 μCi/ml C^14 -labeledL-phenylalanine per well (seeNote 14). - Treatments are maintained for 24 h in incubator at 37C and
5% CO 2 (seeNote 15).
Cell harvest - At the end of the treatment, put dishes on ice.
- Rinse them twice with cold PBS (seeNote 16).
- Add 90μl of lysis buffer and scrap the cells.
- Transfer lysates into 1.5 ml Eppendorf reaction tubes.
- Centrifuge lysates for 15 min at 16,100gat 4C and collect
supernatants. - Protein concentration is measured in supernatants by Lowry
method using BIO-RAD protein assay kit in a 96-well plate.
330 Florence Mailleux et al.