Leucine incoporation in neonatal cardiomyocytes
Reagents:
- Liquid scintillation cocktail from MP Biomedicals, Cat: 882475.
- 3H-Leucine from Perkin Elmer, Cat: NET460A001MC.
- Trichloacetic acid, 10% from LanChem Inc. Cat: LC26230-2.
To measure hypertrophic growth of cardiomyocytes, there are many different approaches including quantification of cell surface area, measuring total protein concentration and RNA concentration. The best and most reliable way is to measure leucine incoporation using radio-labeled 3H-leucine.
1. Day 1, isolate and culture rat neonatal cardiomyocytes.
2. Day 3, starve cardiomyocytes by switching serum-containing medium to serum-free medium.
3. Day 4, start treatment. Dependent on the treatment regime, you may start add 3H-leucine into the medium. I use 1uCi/ml 3H-leucine and use 2 ml medium in each well of 6-well plate.
4. Day 5 or 6, after treatment, when harvesting cells, remove culture medium and wash cells with ice-cold PBS for 3 times. You may need to store all the 3H-leucine containing medium to special containers.
5. Add 2 ml ice-cold 10% TCA in each well and rotate at 4C for 30 min.
6. Remove TCA and wash with ice-cold 95% ethanol for 2 times.
7. Add 1 ml 0.5N NaOH into each well and rotate at 37C to dissolve precipitates for 6 hrs.
8. Add 1 ml 0.5N HCl to neutrolize the solution and transfer all to a scintillation vial.
9. Add 18 ml scintillation cocktail and shake vigorously until the solution becomes clear.
10. Count.