AMPK Methods and Protocols

2. Distribute each intermediate solution for glycolysis assay
   prepared as in Subheading2.3,items 6– 8 , in the sensor car-
   tridge ports as followed:

–80μl of 72.5 mM glucose intermediate solution in the

injection port A.

–80μl of 8.25 μM oligomycin intermediate solution for

glycolysis in the injection port B.

–80μl of 1.537 M 2DG intermediate solution in the injection

port C.

3.5 Cartridge
Loading
for Mitochondrial
Respiration Assay

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


2. Distribute each intermediate solution for mitochondrial respi-
   ration assay prepared as in Subheading2.3,items 9– 11 , in the
   sensor cartridge ports as followed:
   –80μl of 7.25μM oligomycin intermediate solution in the
   injection port A.
   –80μl of 4.125 μM FCCP intermediate solution in the
   injection port B.
   –80μl of 9.25μM Rot/AA intermediate solution in the
   injection port C.

Table 5

Instrument protocol for the glycolytic assay on primary neuronal culture

Basal Glucose Oligomycin 2DG

Inj port A B C

Cycles 3 3 2 5

Mix 2:30 min 2:30 min 2:30 min 2:30 min

Wait 2:00 min 2:00 min 2:00 min 2:00 min

Measure 2:30 min 2:30 min 2:30 min 2:30 min

Table 6

Instrument protocol for the mitochondrial respiratory assay on primary

neuronal culture

Basal Oligomycin FCCP Rotenone/antimycin A

Inj port A B C

Cycles 3 3 3 3

Mix 1:30 min 1:20 min 00:20 min 1:20 min

Wait 2:00 min 1:20 min 00:00 min 1:20 min

Measure 3:00 min 2:00 min 2:00 min 2:00 min

Seahorse Analysis of AMPK-Regulated Metabolic Fluxes 297