AMPK Methods and Protocols

27. After removing distillated water on microscope slides, put 7μl
    of Vectashield mounting medium with DAPI (seeNote 28)in
    the middle of the drawn circle.


28. After removing distillated water from each dish, pick the cov-
    erslip from the corresponding dish, and turn it over on the
    mounting medium droplet.


29. After 15 min at room temperature, the coverslip will become
    immobilized. Microscope slides can be viewed on any
    structured illumination fluorescent microscope, and cell sur-
    face can be quantified on images using AxioVision 4.8
    (or similar) software (Fig.3a–d).

3.5 Evaluation
of NFAT
Transcriptional
Activity

Cell infection

1. Remove full IMDM medium from each well.


2. Rinse cells with PBS.


3. Add 1 ml of IMDM without serum.


4. Infect cardiomyocytes with NFAT luciferase adenovirus at a
   multiplicity of infection (MOI) of 10 orβ-galactosidase adeno-
   virus at similar MOI as control condition (seeNote 29).


5. After 2 h, add 1 ml of full IMDM medium, and incubate cells
   for 24 h in incubator at 37C, 5% CO 2.
   Cell treatment


6. After 24 h, remove the medium and add 2 ml of fresh IMDM
   without serum.


7. After 2 h, treat cells according to the conditions (seeSubhead-
   ing 3.4 for details)


8. Incubate cells for 24 h in incubator at 37C, 5% CO 2.
   Cell lysis


9. Remove the medium and wash the cells once with cold PBS 1.


10. Remove PBS as much as possible.


11. Add 180μlof1CCLR lysis buffer.


12. Shake culture dishes several times to ensure coverage of all cells
    with lysis buffer.


13. Scrape attached cells and transfer lysates in 1.5 ml reaction
    tubes.


14. Vortex tubes for 10–15 s and centrifuge for 90 s at 12,081g
    at 4C.


15. Transfer supernatants to new tubes.


16. Store samples at 80 C or proceed to next section.
    Luciferase activity measurement


17. Program the luminometer with the appropriate delay time (2 s)
    and the appropriate measurement time (10 s).

336 Florence Mailleux et al.