AMPK Methods and Protocols

cells), labeling for Pax7 (muscle stem marker), Ki67 (proliferation

marker) and MyoD (activation marker and commitment into the

myogenic lineage marker) is performed (seeNote 14). You can also

perform desmin labeling to determine fusion of myogenic cells (see

Note 15) or active caspase 3 labeling to test apoptosis induced by

AMPK activators.

1. Wash the 48-wells plate once with 1PBS (seeNote 16).


2. Add 100μl of 4% PFAperwell and incubate for 10 min at RT.


3. Wash wells with 1PBS for 5 min. Repeat three times.


4. Add 100μl of permeabilization bufferperwell and incubate for
   10 min at RT.


5. Wash wells with 1PBS for 5 min. Repeat three times.


6. Add 100μl of saturation bufferperwell and incubate at least
   1 h at RT.


7. Wash wells with 1PBS for 5 min. Repeat three times.


8. Add 80μl of the first antibody mixper well, and incubate
   overnight at 4C in humid chamber.


9. Wash wells with 1PBS for 5 min. Repeat three times.


10. Add 80μl of the second antibody mixperwell, and incubate for
    1hat37C in humid chamber.


11. Wash wells with 1PBS for 5 min. Repeat three times.


12. Add 100μl of the streptavidin mixperwell and incubate for
    45 min at 37C in humid chamber (except for act-casp3
    labeling alone).


13. Wash wells with 1PBS for 5 min. Repeat three times.


14. Add 100μl of the nuclei staining solution; incubate 10–20 s
    at RT.


15. Wash wells with 1PBS for 5 min. Repeat three times.


16. Add 100μl of mounting medium in each well.


17. Store at 4C until analyzed (seeFig. 2a, b ).

4 Notes

1. Matrigel is a solubilized basement membrane preparation
   extracted from the Engelbreth-Holm-Swarm (EHS) mouse
   sarcoma, a tumor rich in extracellular matrix proteins, includ-
   ing laminin (a major component), collagen IV, heparin sulfate
   proteoglycans, entactin/nidogen and a number of growth fac-
   tors, which occur naturally in the EHS tumor. Defrost the
   Matrigel atþ 4 C overnight, aliquot it in chill Eppendorf,
   and freeze again. On the day of your experiment, defrost the

Muscle Stem Cells Fate 547