AMPK Methods and Protocols

Cell Treatment

9. Proceed to AMPK modulation, by using pharmacological
   approaches (seeTable4 for further details). Alternatively it is
   possible to adopt genetic-based approaches for silencing
   AMPK expression (e.g., siRNA) or to induce the expression
   of deregulated forms of AMPK (e.g., constitutive active or
   dominant negative form of AMPK) [18, 19], using expression
   vectors or viral vectors (seeNote 9).

3.2 Cartridge
Hydration (Day Before
the Assay)

1. Open the box containing the sensor cartridge (Fig.1b).


2. Remove the sensor cartridge with the utility plate from the box.


3. Put away the sensor cartridge with the purple support upside
   down on the working bench.


4. Fill the 24 wells of the utility plate with 1 ml of XF Calibrant.


5. Reposition the sensor cartridge with the purple support in the
   utility pate in such a way that the sensors are correctly
   submerged in the XF Calibrant solution.


6. Place the sensor cartridge with the utility plate in an incubator
   without CO 2 at 37C overnight (seeNote 10).

3.3 Assay
Configuration (Day
Before the Assay)

The Seahorse Analyzer and the Seahorse Wave software should be

turned on the day before the assay to allow the machine to reach

37 C. The assay configuration will differ for glycolysis and mito-

chondrial assay as described below. The assay configuration can be

set up at any time before the reading and can be used as a template

for further assays.

1. Open the Wave software.


2. Design a new template, or open a template previously created,
   according to the assay.


3. In the group definition tab, define the injection strategies,
   parameters, assay media, and cell type filling the information
   following the software wizard.


4. Generate group list according to the assay.


5. In the plate map tab, distribute the groups previously defined
   on the plate map displayed on the screen, as well as the back-
   ground well position according to the cell map (seeabove).


6. In the instrument protocol tab, define the injection sequence
   and the cycles of mix, wait, and measure as detailed in Table 5
   for the glycolysis assay and Table6 for the mitochondrial assay.


7. Save the template.

3.4 Cartridge
Loading for Glycolysis
Assay

1. Take the cartridge from the incubator. Remove the purple
   support and place back the cartridge in the utility plate.

296 Claudia Marinangeli et al.