AMPK Methods and Protocols

identified AMP-activated protein kinase (AMPK), the master met-

abolic regulator of the cell, as an important player in MuSC self-

renewal [4]. Thus, manipulating metabolic pathways might aid to

regulate stem cell fate between self-renewal and differentiation.

This chapter provides full information on how to harvest mus-

cles, sort MuSCs, and then study their fate in vitro. Here, we

describe methods for muscle stem cell extraction and culture in

order to investigate the importance of AMPK in metabolic pathway

acting on MuSC fate in a cell-autonomous manner. A selection of a

combination of specific cell surface receptor markers is used to

isolate pure MuSCs via fluorescence-activated cell sorting

(FACS). MuSC pool is defined through positive selection of cells

expressing bothα7-integrin and CD34, combined with negative

selection of CD45 (marker of hematopoietic lineage), CD31

(endothelial cells), and Sca-1 (mesenchymal and hematopoietic

stem cells) [5]. Then, to study MuSC behavior upon modulation

of AMPK activity, the cell population is entirely assessed by a

combination of Pax7 (muscle stem cell marker), Ki67 (proliferation

marker) and MyoD (activation marker and commitment into the

myogenic lineage marker) staining by immunochemistry. MuSC

population can be deciphered in four groups of cells found:

Pax7+Ki67/MyoD
cells are quiescent, Pax7+Ki67/MyoD+cells

are activated, Pax7
Ki67/MyoD+ are committed cells that are

mostly into the myogenic lineage and finally Pax7
Ki67/MyoD


cells.

2 Materials

All culture media and sterile reagents are from Gibco-Life Tech-

nologies and plastics from BD Biosciences, unless otherwise

indicated.

2.1 Mouse
Dissection and MuSC
Extraction

1. Material for mouse dissection (thin forceps, razor blade or thin
   scissors, thin sharp scissors).


2. Shaking water bath setup at 37C.


3. Vortex.


4. Ethanol diluted in water 70% (v/v).


5. Petri dishes 60 mm.


6. 30 ml polystyrene containers.


7. 0.22μm Millex Syringe filter (Millipore, #SLGP033RS) and
   20 ml syringe (TERUMO, #SSþ20ES1)


8. 70μm cell strainers.


9. 30μm CellTrics®strainers.


10. 15 and 50 ml Falcon tubes.


11. Polypropylene round bottom FACS tubes.

540 Marine Theret et al.