- We determined the optimal cell confluence via an optimization
test (Fig.3). As the cell density can vary according to the cell
type to be tested, we strongly advise the reader to run an
optimization test, to determine the optimal quantity of cells
for the experiments to run. - The treatment can last from few minutes, accounting for the
minimal time required for the drugs to efficiently act on AMPK
activity, to several hours, according to the experimental design. - The incubation time can vary between 6 and 72 h, the optimal
being ON. The purple support is designed to avoid dehydra-
tion of the cartridge during the hydration phase, so it is
strongly recommended to leave the support during the entire
hydration time, and remove it only before loading the
cartridge. - If needed, addition of pharmacological modulators of AMPK
can be added during rinsing and incubation steps. - In case cell detachment occurs, we invite the reader to optimize
cell adherence, by using an alternative coating procedure. - The incubation time can be changed according to the cell type.
In some cases, the assay can be run immediately after the
washing steps, while in other cases the incubation time can
reach 1 h. In both cases, we invite the reader to test different
incubation times. The optimal one will be reflected by a stable
OCR value during the acquisition of the basal respiration. In
any case, due to the limited concentration of nutriments in the
medium, absence of serum and CO 2 during the incubation, we
strongly discourage to incubate cells for an excessive period. - The loading and calibration/equilibration of the cartridge can
be performed either before the cell washing and preincubation
step or during the cell preincubation time. In this last case, be
sure to perform steps in Subheadings 3.5 and 3.6 before
the end of the 40 min cells preincubation step. - If cells are detached after the assay, we advise the reader to
change the coating plate procedure. Alternatively, we advise to
reduce the duration of the mix phase during the assay cycles, or
even abrogate it. In this case, increase the wait time, so to allow
the reagents to diffuse correctly in the medium, once injected.
DNA or protein dosage can be realized in order to normalize
the assay. Otherwise, dispose of cells and medium
appropriately. - Figure4 shows an example of glycolytic test run on primary
neuronal culture in presence (Glc), or absence (Ctrl) of glucose
as metabolic substrate. The results show that in absence of
glucose, glycolysis and glycolytic capacity are strongly abro-
gated (Fig.4a and b), confirming that the ECAR monitored is
302 Claudia Marinangeli et al.