AMPK Methods and Protocols

3.2 Measure of MuSC
Proliferation

1. Coat 48-well plate by adding 80μl of coating solution in each
   well and incubate for 30 min at 37C. Wash twice with 1PBS
   to avoid Matrigel lump (seeNote 1).


2. Plate 50 MuSCs (numbers from the sorting)perwell in the
   coated 48-well plate in MuSC growth medium containing
   AMPK activators or not (seeTable1). Appropriate control
   treatment should be done (e.g., DMSO).


3. From day 2 to day 7, under the microscope and with a cell
   counter, calculate the number of cells in each clone and in each
   well to assess MuSC proliferation and the impact of AMPK
   activation or inhibition on it.

3.3 MuSC
Amplification

This method is continuing from Subheading3.1.

1. Coat 12-well plate by adding 500μl of coating solution in each
   well and incubate for 30 min at 37C. Wash twice with 1PBS
   to avoid Matrigel lump (seeNote 1).


2. Plate cells in the coated 12-well plate at 3000 cells/cm²in
   MuSC growth medium.

250K

200K

150K

ABC

DE F

100K

50K

105

104

103

− 103

0

105

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− 103

0

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− 103

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FSC-A FSC-H SSC-H

SSC-A

DAPI

SSC-A FSC-W SSC-W

DAPI

CD34-FITC

CD45/CD31/Sca1-PE a7-integrin-APC

200K 250K

00 50K 100K 150K 200K250K − 103 103 104 105 − 103 0 103 104 105

Live cells

89.0

Lin-

32.1

MuSCs

19.5

Single Cells

Single Cells91.5 86.0

P1

59.6

0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K

250K

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0

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0

Fig. 1Muscle stem cell extraction by cell sorting. Gating strategy to sort muscle stem cells (MuSCs) from hind
limbs. MuSCs are sorted on DAPIneg, CD45/CD31/Sca1negand CD34/α7intposcells. (a) Cells are selected on
their SSC/FSC localization and then (b,c) double selected on low FSC-H and SSC-H to avoid cell doublets. (d)
Cells are then negatively selected for DAPI and (e) CD31/CD45/Sca1. (f) Finally, the MuSC population is the
double positive for CD34 andα7integrin. This last gate is the population that has to be sorted

544 Marine Theret et al.