- Glucose intermediate solution: 72.5 mM glucose. Dilute 20%
glucose stock solution accordingly. The final concentration
during the assay is 10 mM. - Oligomycin intermediate solution for glycolysis: 8.25μM oli-
gomycin. Dilute 10 mM oligomycin stock solution accord-
ingly. The final concentration during the assay is 1μM. - 2DG intermediate solution: 1.537 M 2DG. The final concen-
tration during the assay is 150 mM. Adjust pH to 7.350.05
using NaOH.
Reagent Intermediate Solutions for Mitochondrial Respiration
Assay
To run a mitochondrial respiration assay, solutions of oligomy-
cin FCCP and Rot/AA are calculated and indicated in Table3.
Prepare the following intermediate solution of each reagent in
pre-warmed MGA medium:- Oligomycin intermediate solution for respiration: 7.25μM oli-
gomycin. Dilute 10 mM oligomycin stock solution accord-
ingly. The final concentration during the assay is 1μM. - FCCP intermediate solution: 4.125μM FCCP. Dilute 2.5 mM
FCCP stock solution accordingly. The final concentration dur-
ing the assay is 0.5μM. The optimal FCCP concentration has
been determined from a dose response optimization test (see
Note 4and Fig.2a and b). - Rot/AA: 9.25μM Rot, 9.24μM AA. Dilute 2.5 mM Rot/AA
stock solutions accordingly. The final concentration during the
assay is 1μM.
Table 3
Calculation of intermediate concentration of reagents to be injected to run a mitochondrial assay
Reagent Port Dilution factor [Final] [Stock] [Intermediate] [Final]
Oligomycin A 7.25 2.1 ml 10 mM 7.25μM1μM
FCCP B 8.25 2.1 ml 2.5 mM 4.125μM 0.5μM
Rot/AA C 9.25 2.1 ml 2.5 mM 9.25μM1μMTable 2
Calculation of intermediate concentration of reagents to be injected to run a glycolytic assay
Reagent Injection port Dilution factor Final volume (ml) [Stock] [Intermediate] [Final]
Glucose A 7.25 3 20% 72.5 mM 10 mM
Oligomycin B 8.25 2.1 10 mM 8.25μM1μM
2DG C 9.25 2.1 – 1.537 M 150 mMSeahorse Analysis of AMPK-Regulated Metabolic Fluxes 293