Measurement of Mitochondrial ROS Production - Protein Misfolding and Cellular Stress in Disease and Aging - page 241

Percoll and adjustesd to pH 7.4. This Percoll solution is
diluted with MSEGTA mixture described above to 25%
v/v) and centrifuged at 30,000 × g × 10 min. Purified mito-
chondria fraction was collected at the bottom of the tube,
resuspended in MSEGTA without added BSA and
washed 2 times by centrifuging at 12,000 g × 10 min. Final
mitochondrial pellet was resuspended in MS buffer com-
prising 225 mM mannitol, 75 mM sucrose, 20 mM
HEPES-NaOH, pH 7.4 and stored on ice. Protein content
was estimated by a commercial BCA assay (“Pierce
Biotechnology”/“Thermo Scientific,” USA). Typically
this procedure yields essentially pure, primarily non-synap-
tic, well-coupled mitochondria with respiratory control
index 7-9 on glutamate plus malate.
2. Prepare the base buffer comprising 125 mM KCl, 4 mM
KH 2 PO 4 , 14 mM NaCl, 20 mM HEPES-NaOH, pH 7.2,
1 mM MgCl 2 , and 0.020 mM EGTA, in de-ionized water
(Milli-Q, “Millipore”), adjust pH with KOH and sterilize the
buffer by filtering it through 0.22 mm membrane. Stable for
at least 3 months if kept sterile. Store at room temperature (or
at +4°C) in an air-tight glass container. DO NOT USE ANY
PLASTIC VESSELS TO STORE THE BUFFER! Avoid
freezing the buffer. Prepare 1 ml of 100 mg/ml solution of
bovine serum albumin, essentially fatty acids free, dissolved in
de-ionized water (Milli-Q, “Millipore”); store frozen at
−20°C. This can be frozen and thawed many times. Reconstitute
the complete incubation buffer mixture right before the
experiments; discard unused buffer after the experiment.
(a) Note that the composition of our incubation buffer mim-
ics the ionic composition of cell cytosol and allows one to
study various and many mitochondrial functional activities
under standard, directly comparable experimental condi-
tions. Bovine serum albumin and EGTA are added to
increase the degree of coupling of mitochondria by remov-
ing loosely bound long chain fatty acids and residual cal-
cium contaminating other chemicals, respectively. Although
the composition of incubation buffer can affect the mea-
surements of H 2 O 2 , we have had good experience in using
various other buffers that are commonly used in experi-
ments with isolated mitochondria. For example, simplest
iso-osmotic buffer suitable for H 2 O 2 measurement can be
composed of 225 mM mannitol, 75 mM sucrose, and
20 mM HEPES-NaOH (pH 7.4). Basically, the choice of
a buffer should suit the experimental needs of the study. It
is, however, strongly advised to avoid including reducing
sugars such as glucose, bicarbonate, and compounds that
are substrates or inhibitors of horseradish peroxidase. An
optimal pH range for these measurements is 7.2-7.6.