Dear Manu,
Using Fluorescent Probes
2.3.1 Measurements
2.3.1.1 Measurement of cytosolic ROS production with the use of DHE
Cytosolic superoxide can be measured using the DHE fluorescent probe. Cells
grown in multiwell plates should be incubated for 20 min (needed to allow the
probe to enter the cell and start the reaction) at 37 C in 0.5 ml of DMEM
containing 0.5 mM DHE. After the incubation, cells should be washed twice
with PBS. The fluorescence can be measured in the measurement buffer using
a microplate reader set to 535 nm excitation (Ex bandwidth: 25 nm) and
635 nm emission (Em bandwidth: 35 nm) wavelengths. This is a single-read
measurement that does not define the kinetics of the reaction.
2.3.1.2 Measurement of mitochondrial ROS production with the use of MitoSOX Red
Mitochondrial superoxide production can be measured using the MitoSOX
Red fluorescent probe. Cells grown in multiwell plates should be washed
twice with PBS to remove the medium and subsequently incubated for
10 min (needed to allow the probe to enter the cell and start the reaction
within the mitochondria) at 37 C in 0.5 ml of measurement buffer containing
5 mM MitoSOX Red. After the incubation, the cells should be washed
twice with PBS. The fluorescence can be monitored in the measurement
buffer with a microplate reader set to 510 nm excitation (Ex bandwidth:
10 nm) and 595 nm emission (Em bandwidth: 35 nm) wavelengths. Alternatively,
if the signal is high enough, the fluorescence can also be measured using
400 nm excitation to measure only the superoxide-specific product of
MitoSOX Red oxidation. This is a single-read measurement that does not
define the kinetics of the reaction. Avoid exposing the MitoSOX Red stock
solution and dye-loaded cells to light before measuring.
2.3.1.3 Measurement of ROS production with the use of CM-H2DCFDA
The rate of H2O2 production can be measured using the CM-H2DCFDA
fluorescent probe. Cells grown in multiwell plates should be washed
twice with PBS to remove the medium. Next, add 0.5 ml of measurement
buffer containing 2 mM CM-H2DCFDA. Immediately after addition, start
measuring the fluorescence in kinetic mode at 485 nm excitation (Ex bandwidth:
20 nm) and 520 nm emission (Em bandwidth: 10 nm) wavelengths
http://www.unife.it/dipartimento/medicina-sperimentale-diagnostica/sezioni-centri/sezione-di-patologia-generale/signaltransductionlab/publications/143.pdf
Hoping this will be helpful,
Rafik
Dear Manu,
in addition to Rafik's references, I suggest another fluorescent agent, CellROX Deep Red (see details in Life Technologies site), allowing to eliminate interfering fluorescence if you use such agents for treatments. Moreover, it worked well in our hands to mesure real-time ROS (H2O2) production in living cells by fluorescent microscopy. See the method in this link:
http://www.nature.com/articles/srep21088
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