AMPK Methods and Protocols

the sensor cartridge (Fig.1b and c). For glycolysis assay, glucose,

oligomycin, and 2-deoxy-D-glucose (2DG) will be sequentially

injected, while for mitochondrial respiration assay, oligomycin,

carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP),

and rotenone/antimycin A (Rot/AA) will be serially injected.

In order to obtain the desired final concentration for each

reagent to be injected, appropriate intermediate solution of each

reagent needs to be calculated. This will depend on the volume of

the reagent injected and the final volume of the medium once the

injection has occurred. In the present protocol, the starting volume

of medium in each well is 500μl, while every injection is of 80μl.

In this way, following the A to D injection sequence, dilution of

each injected reagent will be calculated according to Table1. The

present protocol is based on the use of three of the four injection

ports. The remaining port allows the injection of an additional

reagent or drug during the Seahorse XFe assay, further increasing

the possibilities of metabolic tests and strategies that can be

performed.

Reagent Stock Solutions

1. Glucose solution: 20% (w/v) glucose. Prepare a sterile solution
   by dissolving 20 g ofD-glucose in 100 ml of ultrapure water,
   filter (0.22μm), and store at 4C.


2. Oligomycin: 10 mM oligomycin A in DMSO (seeNote 3).


3. FCCP: 2.5 mM FCCP in DMSO. Keep protected from light
   (seeNote 3).


4. Rot: 2.5 mM rotenone (Rot) in DMSO (seeNote 3).


5. AA: 2.5 mM antimycin A (AA) in DMSO (seeNote 3).

Reagent Intermediate Solutions for Glycolysis Assay

To run a glycolysis assay, intermediate solutions of glucose,

oligomycin and 2DG are calculated and indicated in Table2.

Prepare the following intermediate solution of each reagent in

pre-warmed MGA medium:

Table 1
Calculation of the dilution factors for the reagents to be loaded in the cartridge injection ports

Injection port

sequence

Volume (μl) for

each injection

Volume (μl) of medium

after the injection

Dilution factor of each

reagent to be loaded

A 80 580 7.25

B 80 660 8.25

C 80 740 9.25

D 80 820 10.25

292 Claudia Marinangeli et al.