assay, the metabolic measurements are made with a solid state
fluorescent probe attached to a specialised plate, which allows
separate and simultaneous measurements of H
+ and O 2 in each
well of the plate. Each probe is surrounded by four injection
ports, which are loaded with assay-specific inhibitors and programmed to be added to the cells at precise times during the assay
in order to assess metabolic function. On the day before the assay,
the probe plates are rehydrated in sterile water at 37
C in atmospheric oxygen. On the day of the assay the inhibitors are added to
their respective ports in preparation for the assay and loaded onto
the Seahorse machine. The inhibitors used in the Mito Stress Test
are Oligomycin, which blocks ATP synthase of electron transport
chain (ETC) and causes a decrease in oxygen consumed via
OXPHOS and usually an increase in glycolysis; FCCP (Carbonyl
cyanide-4 (trifluoromethoxy) phenylhydrazone), an uncoupling
agent that disrupts the mitochondrial membrane potential by collapsing the proton gradient; and a mixture of Rotenone, a complex I
inhibitor of ETC, and Antimycin A, a complex III inhibitor of ETC
to inhibit OXPHOS. Following the assay, the plates are removed
and data is normalised by the immediate measurement of cell
number, DNA or protein content. It is important to consider
bioenergetics in the context of cellular mitochondrial mass, which
may differ between treatments or phenotypes. A double-staining
method for evaluating mitochondrial content and activity is
described in this protocol.
3.2 Day 1 – Seeding
of the Cells and Probe
Rehydration
1. In a laminar flow hood, hydrate the XF assay cartridge plate.
Lift the green probe cartridge from the utility plate and place it
with probes facing up to avoid damage.
2. Fill each well of the utility plate with 200 μL of ultrapure water
and replace the probe cartridge, submerging the sensor sleeves
thoroughly in the solution.
3. Place the cartridge at 37
C in a non-CO 2 incubator overnight
along with a falcon tube containing 20 mL of sterile calibration
solution in preparation for the following day.
4. Coat the cell culture plate (See Note 2). Cover the bottom of
the plate with matrix and incubate at room temperature for
1–3 h.
5. Rinse the wells at least twice with sterile PBS using a multichannel pipette and reservoir. Allow wells to dry in the laminar
flow hood.
6. Select the appropriate cell density. As cells have different proliferation capacities, the number of cells per well should be determined empirically for each cell line. Cells should be seeded so
that all the wells are 80–90% confluent on Day 2.
Studying EMP Bioenergetic Switch Using Seahorse Analyzer
331
fluorescent probe attached to a specialised plate, which allows
separate and simultaneous measurements of H
+ and O 2 in each
well of the plate. Each probe is surrounded by four injection
ports, which are loaded with assay-specific inhibitors and programmed to be added to the cells at precise times during the assay
in order to assess metabolic function. On the day before the assay,
the probe plates are rehydrated in sterile water at 37
C in atmospheric oxygen. On the day of the assay the inhibitors are added to
their respective ports in preparation for the assay and loaded onto
the Seahorse machine. The inhibitors used in the Mito Stress Test
are Oligomycin, which blocks ATP synthase of electron transport
chain (ETC) and causes a decrease in oxygen consumed via
OXPHOS and usually an increase in glycolysis; FCCP (Carbonyl
cyanide-4 (trifluoromethoxy) phenylhydrazone), an uncoupling
agent that disrupts the mitochondrial membrane potential by collapsing the proton gradient; and a mixture of Rotenone, a complex I
inhibitor of ETC, and Antimycin A, a complex III inhibitor of ETC
to inhibit OXPHOS. Following the assay, the plates are removed
and data is normalised by the immediate measurement of cell
number, DNA or protein content. It is important to consider
bioenergetics in the context of cellular mitochondrial mass, which
may differ between treatments or phenotypes. A double-staining
method for evaluating mitochondrial content and activity is
described in this protocol.
3.2 Day 1 – Seeding
of the Cells and Probe
Rehydration
1. In a laminar flow hood, hydrate the XF assay cartridge plate.
Lift the green probe cartridge from the utility plate and place it
with probes facing up to avoid damage.
2. Fill each well of the utility plate with 200 μL of ultrapure water
and replace the probe cartridge, submerging the sensor sleeves
thoroughly in the solution.
3. Place the cartridge at 37
C in a non-CO 2 incubator overnight
along with a falcon tube containing 20 mL of sterile calibration
solution in preparation for the following day.
4. Coat the cell culture plate (See Note 2). Cover the bottom of
the plate with matrix and incubate at room temperature for
1–3 h.
5. Rinse the wells at least twice with sterile PBS using a multichannel pipette and reservoir. Allow wells to dry in the laminar
flow hood.
6. Select the appropriate cell density. As cells have different proliferation capacities, the number of cells per well should be determined empirically for each cell line. Cells should be seeded so
that all the wells are 80–90% confluent on Day 2.
Studying EMP Bioenergetic Switch Using Seahorse Analyzer
331