Measurement of Mitochondrial ROS Production - Protein Misfolding and Cellular Stress in Disease and Aging - page 242

3. Prepare 10 mM stock solution by adding 330 ml of anhydrous
DMSO to the content of 1 mg package. Wrap in foil to pro-
tect from light. Aliquot by 10-20 ml and keep frozen at
−80°C; an aliquot can normally be thawed-re-frozen once or
twice. Discard when a faint pink color appears.
4. Prepare the stock solution by dissolving 1,000 U of horseradish
peroxidase in 1 ml of de-ionized water (Milli-Q, “Millipore”).
Aliquot by 20-50 ml and keep frozen at -80°C; discard unused
solution after the experiment (do not re-use).
5. For all aliquoted reagents, use only non-colored, clear plastic
tubes (e.g. PCR tubes or Eppendorf ™ type 1.7 ml tubes), or
- whenever possible - glass tubes.
6. To note, de-energized mitochondria do not routinely produce
ROS. It is therefore suggested to have ready at least some com-
mon mitochondrial respiratory substrates and respiratory chain
inhibitors, although the exact set depends on the purpose and
the design of the ROS assay. In either case, some standard sub-
strates and inhibitors can come up handy, such as (stock solu-
tions) 1 M sodium glutamate, 1 M sodium malate, 1 M sodium
succinate, all prepared in de-ionized water; also 1 mg/ml
Antimycin A and 1 mM rotenone, prepared in absolute ethanol.
These reagents can be frozen-thawed multiple times.
7. Prepare small amount (~250 ml) of ~40,000 U/ml stock
solution in de-ionized water (Milli-Q, “Millipore”) right
before the experiment. Do not freeze; keep on ice protected
from light. Discard unused solution after the experiment.
8. Prepare 1 M solution in de-ionized water (Milli-Q,
“Millipore”). Store frozen.
9. Prepare the stock solution by dissolving 10,000 U of super-
oxide dismutase in 1 ml of de-ionized water (Milli-Q,
“Millipore”). Aliquot by 20-40 ml and keep frozen at −80°C;
discard unused solution after the experiment (do not re-use).
10. In theory, a spectrophotometer can be used to detect ROS, as
the compound produced in peroxidase catalyzed oxidation of
Amplex Red Ultra by H 2 O 2 has large absorption coefficient
(please contact “Invitrogen” company for details). To the
best of our knowledge, there were no publications where
mitochondrial ROS were detected with Amplex Red Ultra
spectrophotometrically. In either case, all the steps, reagents,
and other information presented in this manuscript remain
relevant irrespectively of whether a fluorimeter or a spectro-
photometer is used to detect ROS.
11. DO NOT USE METAL-CONTAINING DEVICES (e.g.
Hamilton syringes with steel needles) to handle H 2 O 2 solutions,
substrate solutions, inhibitors, mitochondria or other liquids.