MCDB-131 medium or KB cells (10^7 cells/ml) in RPMI 1640
medium, as previously reported [16, 17]). After ~30 s of
equilibration, the basalJO^2 can start to be recorded until
reaching a steady state. Although their respective concentra-
tions need sometimes to be adjusted depending of the cell type,
inhibitors and substrates can next be added to the cells in order
to assess the mitochondrial respiratory rates in various states, as
described in Subheading3.1 for intact hepatocytes.
- Glucose can be replaced (or complemented) by various exoge-
nous substrates such as lactate/pyruvate (20/2 mM), glycerol
(20 mM), octanoate (4 mM), or BSA-palmitate (2 mM), as
previously reported [15, 18].
- CCCP can be replaced by DNP and used at the final concen-
tration of 150μM.
- Antimycin can be replaced by myxothiazol and used at the final
concentration of 4μM.
- http://www.agilent.com/en-us/solutions/cell-metabolism-
(seahorse).
- The step-by-step addition of oligomycin, CCCP, and antimy-
cin/rotenone every 15–20 min allows to assess the mitochon-
drial respiration coupled to ATP synthesis (oligomycin-
sensitiveJO 2 ), the proton leak (oligomycin-insensitiveJO 2 ),
the maximal respiratory rate in uncoupled state (or spare respi-
ratory capacity, SRC), and the non-mitochondrial respiration
(antimycin/rotenone-insensitiveJO 2 ), respectively.
- When using succinate/malate as substrate, add 2.5μl of rote-
none (stock: 2 mM; final concentration: 2.5μM) in order to
prevent reverse flux of electrons through the respiratory-chain
complex I.
- The use of anesthetics should be avoided because they modify
mitochondrial functions.
- Mitochondria can also be prepared from cells according to the
same method than the one described in Subheading 3.3.
Briefly, cells from culture dishes (~10 millions) are washed
once with PBS and then scraped in 2 ml of ice-cold homogeni-
zation buffer. After homogenization in an ice-cold 10-ml
Potter-Elvehjem (~20 ups and downs on ice at 500 rpm), the
suspension is transferred in a syringe and passed through a
25 G needle in order to completely disrupt the cells. The
subsequent centrifugation steps and final resuspension are sim-
ilar to the ones described for mitochondria isolation from
whole tissue in Subheading3.3.
- Experiments are carried out at 37C, but the temperature can
also be lowered to 30C for prolonged use owing to mem-
brane fragility of isolated mitochondria.
Methods for Assessing Mitochondrial OXPHOS 285
