AMPK Methods and Protocols

21. The luminometer measures the light produced for a period of
    10 s. The plate is advanced to the next well for light measure-
    ment (Fig.4a).


22. Wash the luminometer injector with distillated water.

3.6 Evaluation ERK1/
2 Signaling Pathway

Such evaluation is performed on cardiomyocytes cultured as

described in Subheading3.3 except that incubation time with PE

and AMPK activator phenformin is restricted to 1 h (seeNotes 22

and 31 ) (Fig.4b and c).

4 Notes

1. Trypsin (Gibco, ref.: 27250-018) is sensitive to heat and is
   activated at 37C.


2. Collagenase (Gibco, ref.: 17101-015) is activated at 37C.
   Prepare a solution of 80 ml for 60 rat pups. Filter the solution
   and divide into eight aliquots of 8–10 ml in 15 ml tubes.


3. Protect RBC lysis buffer from the light.


4. Prepare lysis buffer freshly and stored on ice until use.


5. This adenoviral construction contains nine multimerized
   NFAT binding sites upstream of a minimal TATA-containing
   promoter fused to luciferase. It allows an efficient evaluation of
   NFAT transcriptional activity in primary cultures.


6. Equilibrate luciferase assay reagent at room temperature.


7. This step can be done by inverting tubes. Some pellets can be
   not perfectly stuck on the bottom of the tube; do not hesitate
   to let a small amount of supernatant to prevent loss of
   cardiomyocytes.


8. Set the tube in a horizontal position as much as possible, place
   the pipette perpendicularly to wall tube, and gently add the
   solution in order to make two separate layers, one under with
   the Percoll gradient solution and one above with the cardio-
   myocyte solution.


9. The band in the middle of the tube contains endothelial cells,
   fibroblasts, and other cell fragments.


10. This step can be performed during the centrifugation of Percoll
    gradient solution.


11. This step can be performed during red blood cell lysis.


12. Make the mean of three squares in burker chamber. Do not
    take into account the blue cells which are dead cells. Cell
    concentration in stock solution (per ml)¼number of cells/
    square multiplied by 10,000 and by 10 (corresponding of the
    dilution in trypan blue).

338 Florence Mailleux et al.