AMPK Methods and Protocols

3. Change the media every day during 4 days (rinse once with
   DMEM/F-12 and then add fresh MuSC growth medium) and
   then every 2 days until the cells entirely recover the well (from
   4 to 7 days).


4. Harvest the cells by washing the well once with 1PBS. Then,
   incubate the plate with 100μl of 0.25% trypsin EDTA at 37C
   for 1 min. Check cell detachment under a microscope. Stop cell
   detachment by adding 200μl of FBS. Harvest and spin cells at
   room temperature (RT) at 350gfor 7 min. Resuspend the
   pellet in 1 ml of MuSC growth medium.


5. Count the cells using a hemocytometer, a cell counted and
   viability dye (e.g., trypan blue).


6. Plate cells in 48-well plate coated with Matrigel (seeNote 1)at
   30,000 cells/cm²in MuSC growth medium (seeNotes 12and
   13 ).


7. Six hours after plating, perform Pax7/Ki67/MyoD labeling to
   check MuSC activation (seeSubheading3.5).

3.4 Activation/
Inhibition of AMPK
in MuSCs

1. Six hours after plating, switch the media for MuSC differentia-
   tion medium, containing AMPK activators or not (seeTable1).
   Appropriate control should be done (e.g., DMSO or water).


2. Forty-eight hours after switching the media, check that your
   cells form myotubes and perform Pax7Ki67MyoD labeling (see
   Subheading3.5 and Fig.2). For all molecules and concentra-
   tions, you should check that apoptosis is not highly induced.
   To proceed, perform an immunolabeling against the active
   form of caspase 3 (seeSubheading3.5).

3.5 Immunolabeling
of MuSCs

To determine the percentage of Pax7+Ki67/MyoD
(quiescent

cells), Pax7+Ki67/MyoD+ (activated cells), Pax7
Ki67/MyoD+

(differentiating cells) and Pax7
Ki67/MyoD
cells (differentiated

Table 1

List of AMPK activators or inhibitor and their range of concentration to be

used in vitro

Molecules

Concentration range

(min–max)

5-aminoimidazole-4-carboxamide-1-β-D-

ribofuranoside (AICAR)

0.1–1 mM

Compound 991 0.1–10μM

2 deoxy-D-glucose (2-DG) 50–500 nM

STO-609 1–10μM

Muscle Stem Cells Fate 545