AMPK Methods and Protocols

3.6 Equilibration 1. Open the assay template previously configured (seeSubheading
3.3) on the Seahorse Wave software.

2. Start the running of the assay by clicking on the run assay
   button on the screen.


3. Place the sensor cartridge with the utility plate on the machine
   tray taking care of the cartridge orientation (the notch needs to
   be placed on the bottom left).


4. Confirm the presence of the cartridge to the software by click-
   ing the confirmation button displayed on the screen.


5. Let the calibration and equilibration step to run.

3.7 Cells Washing
and Preincubation
in Assay Medium

1. Take the cell plate out of the incubator and check the state of
   the cells under a microscope to verify cell viability, morphology,
   and confluence.


2. Remove the medium from the each well leaving around 50μlof
   medium to avoid cells from drying during the washing steps.


3. Add 650μl of pre-warmed MGA or MRA at 37C to each well
   (for glycolysis or mitochondrial respiration assay, respectively).


4. Repeatsteps 2and 3 two more times.


5. Replace entirely the medium with 500μl of pre-warmed MGA
   or MRA at 37C well by well (for glycolysis or mitochondrial
   respiration assay, respectively) (seeNote 11).


6. Ensure cell viability and confluence under the microscope
   (make sure cells did not detach) (seeNote 12).

Incubate the cell plate at 37C in an incubator without CO 2

for 40 min. This incubation time is required for the equilibration of

the medium with the external environment and the stabilization of

cell metabolism after the washing steps (seeNotes 13and 14 ).

3.8 Plate Reading 1. Once the calibration is over, remove the cell plate from the
incubator and confirm the opening of the tray by clicking on
the button displayed on the screen.

2. Substitute the utility plate with the cell plate on the tray.
   Confirm the presence of the cell plate by clicking on the button
   displayed on the screen. The assay reading should start
   automatically.


3. At the end of the reading, confirm the discard of the cartridge
   and cell plate on the screen. Empty the tray and discard the
   cartridge in the appropriate waste disposal. Take out the cell
   plate, check the presence of the cells, remove the media, and
   store the cell plate at 20 C if further analyses are needed (see
   Note 15).

298 Claudia Marinangeli et al.