AMPK Methods and Protocols

capacity. Due to their inhibiting action on the complex of the

electron transport chain, the addition of Rot/AA totally inhibits

the OCR. The residual OCR is determined by non-mitochondrial

reactions occurring in the cells (seeNote 17). Besides glucose

metabolism, with the Seahorse XFe respirometry assay, it is possible

to test alternative substrates, such as lactate and ketone bodies

(Fig.5 andseeNote 18).

4 Notes

1. Do not overheat the medium. The medium should be prepared
   freshly each time. Medium might be stored at 4C for no
   longer than 48 h.


2. Alternatively, sodium pyruvate, L-lactate, ketone bodies, or
   fatty acids can be used to assess the mitochondrial capacity to
   use alternative substrates to glucose.


3. For every reagent resuspended in DMSO, we recommend
   storage at 20 C in single use aliquots to avoid freezing/
   thawing cycles.


4. The optimization test is required to determine the optimal
   FCCP concentration to inject during mitochondrial test,
   which can vary according to the cells type and confluence.
   For this step it is possible to inject FCCP once or twice and
   to verify if the effect of the first injection can still be increased.
   In this case, it is of crucial importance to calculate correctly the
   final concentration of FCCP in the wells upon the second
   injection.


5. The plate coating described here has been optimized in our
   laboratory for primary neuronal culture. However, we propose
   to the reader to use the coating procedure used in his
   laboratory.


6. The area of a Seahorse XFe24 well is equivalent to a well of a
   96 well plate.


7. We invite the reader to proceed with his own protocol to obtain
   isolated cells, but for further details on the procedure, please
   refer to Domise et al. [17].

ä

Fig. 5(continued) substrates and in response to the sequential injection of 1μM oligomycin, 0.5μM FCCP,
and 1μM Rot/AA, as indicated byarrows.(b,e) Basal respiration calculated as the difference between OCR
before oligomycin injection and OCR after Rot/AA addition. (h,k) OCR increase upon the injection ofβHBA or
AcAc. (c,f,i,l) Maximal respiration expressed as the difference between OCR after FCCP and Rot/AA injection.
Cells were tested between 14 and 17 DIV. Data are meanSD ofn¼3–5. Unpairedt-test, **p<0.05,
***p<0.01

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