gradient, causes OCR to reach a maximum, as mitochondria
are no longer limited by the supply of ADP. However, prior
addition of phenformin, a potent AMPK activator, will reduce
OCR and prevent any DNP-induced increases in OCR, con-
firming that it is an inhibitor of the ETC rather than the F1Fo
ATP synthase. As with cell number, it is important to titer the
concentration of compounds to achieve the maximum effects.
For HEK293 cells, we find that 10 mM phenformin and
100 μM DNP are sufficient to maximally reduce and stimulate
OCR, respectively.- We routinely use phenformin for this purpose.
- The assay protocol is determined by a number of parameters,
including the number of injections to be performed and the
expected OCRs for different cell types. Consult the manufac-
turer’s guidelines to set these. For HEK293 cells, we typically
perform the following steps: mix for 2 min and rest for 10 min.
To establish a baseline of oxygen consumption rate, we take
three measurements as follows: mix for 2 min, rest for 2 min,
and measure for 2.5 min. These measurement cycles are then
repeated as desired, following each compound injection. To
accurately determine the effects of a drug, the OCR should
always be allowed to reach a new steady state following injec-
tion. The Seahorse allows for the protocol to be changed while
the assay is in progress, so adjustments can be made if the OCR
is still changing following injection. - To determine the effect of a compound on OCR, we found
it helpful to express the effects as a percentage change in
basal OCR following injection. This normalization removes
the need to determine the absolute numbers of cells in
each well. - The commercially available pACC antibodies appear to recog-
nize both ACC1 phosphorylated on Ser79 and ACC2 phos-
phorylated on Ser212 (mouse numbering). These two
isoforms differ slightly in size (ACC1 265 kDa, ACC2
276 kDa, mouse sequences), and in tissues expressing both
(e.g., mouse liver), it should be possible to resolve them.
Acknowledgments
Studies in the Hardie Laboratory were supported by a Senior
Investigator Award (097726) from the Wellcome Trust and by a
Programme Grant (C37030/A15101) from the Cancer Research
UK.252 Simon A. Hawley et al.