3.1.2 Seahorse The regulation of mitochondrial oxidative phosphorylation
(OXPHOS) can be assessed in intact adherent cells (e.g., primary
hepatocytes seeded in collagen-coated 96-well plate) using the
newly developed oxygen-sensing fluorophore-based Seahorse tech-
nology (Fig.3). It is worth mentioning that a complete step-by-
step procedure for running a Seahorse assay was recently described
in details elsewhere for mouse/human dendritic cells [7] and could
be used as general guidelines, together with the one provided by
the manufacturer (seeNote 9).
- Add 60μl of collagen type I (4 mg/ml) in 5 ml of 30%
ethanol and pipette 50μl of this intermediate solution in each
well of a Seahorse 96-well plate. Let evaporate at room
temperature in a tissue culture hood. Wash twice with PBS.
Air-dry the plate and expose to UV light for ~10 min. The plate
can be kept for ~1 week at 4C in sterile conditions (sealed
with parafilm). - Hydrate the Seahorse XF cartridge the day before the experi-
ment according to the manufacturer’s guidelines. - Seed 6 104 freshly isolated primary hepatocytes per well in a
Seahorse 96-well plate coated with collagen and containing
200 μl of M199 complete medium for at least 4 h. - Load the injection ports of the assay cartridge with 20μl of the
following chemicals:- Port A (oligomycin): prepare 2.4 ml of intermediate solu-
tion by adding 43μl of oligomycin (stock: 1 mg/ml; final
concentration: 1μg/ml) in 2357μl of 0% FCS/XF assay
medium.
- Port A (oligomycin): prepare 2.4 ml of intermediate solu-
Fig. 3Determination of mitochondrial respiration in adherent cells using oxygen-sensing fluorophore-based
Seahorse™technology. The oxygen consumption rates can be monitored in adherent cells (~e.g., 6.10^3
primary hepatocytes per well coated with collagen; (a) using oxygen-sensing fluorophore-based Seahorse™
technology (b) allowing continuous and simultaneous measurement of oxygen concentration in 24 or 96 wells.
The computer-controlled administration of up to four respiratory-chain inhibitors/activators (c) allows to
dissect mitochondrial bioenergetics, as described in Fig. 2
280 Guillaume Vial and Bruno Guigas