AMPK Methods and Protocols

solution in culture medium by adding 10μL of 10 mM stock

oligomycin into 990 μL of culture medium (this 100-fold

dilution leads to the 100 μM oligomycin stock solution).

Dilute the DMSO solvent in parallel (10μLþ 990 μLof

culture medium) in a separate tube. Diluted stocks can be

kept at 4C and used for up to 1 month.

3. 6μm-polystyrene beads (seeNote 1).


4. Fluorescent-coupled monoclonal antibodies: anti-mouse TcR-
   β-PerCP-Cy5.5; anti-CD4-PECy7; anti-CD8-PE; anti-
   CD45R-FITC.


5. Anti-mouse CD16/32: 1/200 (seeNote 2).


6. Near-IR viability dye.


7. Petri dishes.


8. 15 mL tubes.


9. 5 mL cytometer-adapted tubes.


10. 70μm nylon mesh cell strainer.


11. Flow cytometer.


12. FlowJo (or any other) cell analysis software.

2.3 Mice 1. T lymphocyte-conditional AMPKα1-KO mice (AMPKα^1 flox/
flox-CD4-CRE+)(seeNotes 3and 4 ).

2. Control mice: AMPKα 1 +/+-CD4-CRE+or AMPKα 1 flox/flox-
   CD4-CRE. Wild-type C57BL/6 mice can also be used.

3 Methods

3.1 Validation
of the Bead-Based Cell
Counting Method
(Fig.1)

1. Collect the spleen of a mouse, and mechanically disrupt it with
   the wide side of a syringe piston, in a 5 mL culture Petri dish
   containing 1 mL of red blood cell lysis solution.


2. After 1 min, stop the lysing reaction by adding 10 mL of cell
   culture medium.


3. Filter the cell suspension using a 70μm nylon mesh cell strainer
   on top of a 15 mL tube, and centrifuge at 400gfor 5–10 min
   at 4C.


4. Discard supernatant and suspend the pellet in 10 mL of culture
   medium.


5. Prepare five cytometer tubes containing twofold serial dilutions
   of cells:
   (a) The first tube will receive 500μL of the cell suspension.
   (b) The four other tubes will receive 250 μL of culture
   medium.

470 Se ́bastien Denanglaire et al.