AMPK Methods and Protocols

3.7 Analysis
of Cellular Oxygen
Uptake Using
an Extracellular Flux
Analyzer

The protocol below assumes the use of a Seahorse XF24 instru-

ment. The use of a different analyzer (e.g., XF96) will require the

cell numbers and volumes to be adjusted appropriately, based on

the well size.

1. Harvest a healthy culture of cells, wash in pre-warmed growth
   medium to remove trypsin, and then seed an appropriate num-
   ber of cells into each well of a Seahorse XF Cell Culture
   Microplate (seeNote 11). Cells should be dispensed in 100μl
   of medium (seeNote 12). We recommend seeding cells in
   triplicate for each condition. Add an equal volume of medium
   to control wells that lack cells.


2. Leave cells at room temperature in the tissue culture hood for
   1h(seeNote 13).


3. Place cells in a 37C tissue culture incubator until they become
   adherent.


4. Add 150μl of growth medium to each well, including controls,
   and return the cell plate to the incubator overnight.


5. Remove the XF Sensor Cartridge from the utility plate (do not
   place it facedown as this could damage the sensors). Add 1 ml
   of Seahorse XF Calibrant solution to each well of the utility
   plate and then place the sensor cartridge back on the plate,
   ensuring that the sensors are fully submerged in calibrant solu-
   tion. Place in a humidified, non-CO 2 ,37C incubator
   overnight.


6. Prepare XF Assay Medium. To the XF Base Medium, add
   appropriate amounts of glucose, sodium pyruvate, and gluta-
   mine. Use the same concentrations as normally used in growth
   medium (seeNote 14). Adjust the pH to 7.4 and filter sterilize.
   Media can be stored at 4C.


7. Aspirate all but 50μl of the media from the cells with a
   micropipette, taking care not to disturb the cell monolayer.
   Wash the cells twice in 1 ml of pre-warmed XF Assay Medium
   and add an appropriate final volume of Assay Medium (seeNote
   15 ). Check cell adhesion and culture purity by examining with
   a microscope.


8. Place the cell plate in the 37C non-CO 2 incubator for 1 h.


9. Prepare compounds for injection (seeNote 16). Compounds
   should be prepared at an appropriate concentration in XF Assay
   Medium and the pH adjusted to 7.4 as required. It is important
   to include a positive control compound that is expected to alter
   OCR (seeNote 17) as well as a vehicle control (e.g., DMSO).


10. Load the ports of the sensor cartridge with 75μl of prepared
    compounds. To ensure even injections across all wells, each
    series of port (e.g., all “A” ports, all “B” ports) should have the

248 Simon A. Hawley et al.