- Wash carefully the oxygraph chamber after each measure for
three times with mQ water, then three times with 70% ethanol
and again three times with mQ water, in order to remove any
remaining traces of inhibitors.
3.4 Calculations The oxygen consumption rates (JO 2 ) are calculated by measuring
theΔ[O 2 ]/Δ(t), i.e. the slope of the linear regression of oxygen
concentration (in nmol O 2 ) versus time (in min), and expressed
toward the number of cells (intact or permeabilized cells) or
amount of proteins (isolated mitochondria) placed in the oxygraph
chamber. Of note, the antimycin-sensitiveJO 2 , which corresponds
to mitochondrial respiration, is systematically calculated by sub-
tracting the antimycin-insensitive (non-mitochondrial)JO 2 from
theJO 2 measured at each state. As an example,Δ[O 2 ] at steady
state between min 2 and min 4 post addition of CCCP
(Δ(t))¼240 nmol O 2 ;soJO 2 ¼240/2¼120 nmol O 2 /min. If
the 2-ml chamber contains mitochondria at a concentration of
1 mg protein/ml, the finalJO 2 will be: 120/2¼60 nmol O 2 /
min/mg protein.
4 Notes
- Primary hepatocytes are isolated from rat, mouse, or human
liver using the collagenase method, as previously described
[14–16]. - Krebs-Ringer bicarbonate buffer can also be used instead of
Krebs-HEPES buffer by lowering NaCl concentration to
120 mM and replacing HEPES with 24 mM NaHCO 3. - The dissolution of oxygen varies with temperature and the
medium composition and should therefore be determined
beforehand. - ~15 mg of dry hepatocytes corresponds to ~1 million
cells [17]. - MitochondrialJO 2 can also be measured in cultured cells that
are either in suspension or adherent to plates, dishes, or flasks.
Once collected by low-speed centrifugation (1200 gfor
2 min) or after trypsinization, cells are resuspended at high
concentration in a small volume of fresh culture medium with-
out FBS or antibiotics (or eventually in Krebs-HEPES buffer),
counted and kept on ice until use. For measurement, 1 ml of
cell suspension is transfered into the oxygraph chamber con-
taining 1 ml of culture medium or Krebs-HEPES buffer and,
eventually, exogenous substrates. Of note, the culture medium
and cell number should be adjusted depending on the cell type
and its respiratory rate (e.g., HMEC-1 cells (10^7 cells/ml) in
284 Guillaume Vial and Bruno Guigas