2.1 Equipment 1. Seahorse XFe24 Analyzer.
- Seahorse XFe24 cell culture plates and corresponding sensor
cartridges (Fig.1a and b). - Incubator without CO 2.
2.2 Buffer and Media 1. XF Calibrant (by Seahorse Bioscience). Store at room
temperature.
- Medium for glycolysis assay (MGA): Dulbecco’s Modified
Eagle Medium Base (DMEM without glucose,L-glutamine,
sodium bicarbonate, sodium pyruvate and phenol red, Sigma
cat # D-5030), supplemented with 31.6 mM NaCl, 42,3μM
phenol red, and 20 mML-glutamine. Bring to 37C while
stirring. Adjust the pH at 7.350.05 using 1 M NaOH when
the solution reaches 37C(seeNote 1). - Medium for mitochondrial respiration assay (MRA): MGA
containing 10 mM glucose (seeNote 2). Adjust the pH at
7.350.05 using NaOH or HCl when the solution reaches
37 C.
2.3 Assay Reagents During the assay it is possible to modulate glycolytic or mitochon-
drial fluxes via the addition in the cell medium of up to four
different reagents via the four injection ports (A, B, C, and D) on
Fig. 1Picture of Seahorse XFe24 cell plate, cartridge, and port configuration. (a) Picture of a Seahorse XFe24
cell culture plate. (b) Picture of the Seahorse XFe24 sensor cartridge, indicated by theupper arrow, and the
utility plate, indicated by thelower arrow. The injection ports corresponding to each well are indicated by a
circle.(c) Schematic representation of the injection ports configuration (a–d)
Seahorse Analysis of AMPK-Regulated Metabolic Fluxes 291