5.4. Analysis of ROS generation by flow fluorocytometry or time-lapse
microscopy
The generation of reactive oxygen species (ROS) is typically observed
in several forms of cellular stress and is known to modulate
signal transduction pathways [53]. The effect of ROS generation depends
on several factors, such as subcellular localization and the
amount and the type of ROS. Dihydrorhodamine 123 (MW
346.4 Da) and CM-H2DCFDA (MW 577.8 Da) are two commonly
used general ROS indicators. DHR123 is an uncharged and nonfluorescent
ROS indicator that can passively diffuse across membranes,
where it is oxidized to cationic rhodamine 123, which localizes in
the mitochondria and exhibits green fluorescence. CM-H2DCFDA is
a chloromethyl derivative of H2DCFDA, but it is much better retained
in live cells than H2DCFDA. CM-H2DCFDA passively diffuses
into cells, where its acetate groups are cleaved by intracellular
esterases and its thiol-reactive chloromethyl group reacts with
intracellular glutathione and other thiols. Subsequent oxidation
yields a fluorescent adduct that is trapped inside the cell, which
facilitates long-term studies. Several new indicators of ROS generation
are now available (Molecular Probes, Invitrogen).