AMPK Methods and Protocols

4. Save the results. The results can be exported in Prism format
   for further analysis. Alternatively the results can be exported in
   Excel format and analyzed using dedicated programs, realized
   and released by Seahorse (report generators).

3.9 Glycolytic Test
Analysis

During the glycolytic test, glucose, oligomycin, and 2DG are sub-

sequently injected (Fig.4a). Due to the absence of glucose in the

MGA, the ECAR recorded is dependent on intracellular

non-glycolytic processes. Upon the injection of glucose, glycolysis

is stimulated, causing an increase of ECAR (Fig.4b). The following

addition of oligomycin maximizes the glycolytic flux (glycolytic

capacity), causing a strong increase of ECAR (Fig.4c). This effect

is due to a metabolic compensatory system, in response to the

inhibition of mitochondrial respiration induced by oligomycin

treatment. Finally, 2DG will inhibit glycolysis, thus causing a

decrease of ECAR to the non-glycolytic values (seeNote 16).

3.10 Mitochondrial
Respiration Test
Analysis

In this assay, oligomycin, FCCP, and Rot/AA are subsequently

injected (Fig.5a). Initially the OCR measured is dependent on

mitochondrial respiration at the basal state of the cells (Fig.5b).

The addition of oligomycin, by inhibiting the ATP synthase com-

plex, causes a reduction of OCR. The extent of this effect reflects

the oxygen consumption dedicated to the production of ATP (ATP

turnover). FCCP maximizes the mitochondrial respiration by dis-

rupting the proton gradient between the intermembrane space and

the mitochondrial matrix. The maximal OCR value reached reflects

the maximal respiratory capacity (Fig. 5c), and the difference

between this pick and the basal respiration is the spare respiratory

Fig. 4Glycolytic flux analysis in the presence of glucose as energy substrate. (a) Glycolytic flux in primary
neuronal culture monitored upon sequential injection of 10 mM glucose (Glc), or medium in the control group
(Ctrl), 1μM oligomycin (oligo), and 150 mM 2DG. (b) Glycolysis calculated as the difference in ECAR values
before and after glucose injection. (c) Glycolytic reserve calculated as the difference between maximal ECAR
value after oligomycin, and maximal ECAR value after glucose injection. Cells were tested between 14 and
17 DIV. Data are meanSD ofn¼5. Unpairedt-test, **p<0.05, ***p<0.01

Seahorse Analysis of AMPK-Regulated Metabolic Fluxes 299