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Reactive species/Oxidative stress
All respiring organisms generate in their metabolism reactive oxygen species (ROS)
which may be damaging for cell function. Failure of physiological antioxidant defense or
accumulation of ROS leads to oxidative stress that may be quantified following the reaction of
reactive species or antioxidant molecules within the cell with fluorogenic substrates.
Two indicators have been used to date to study oxidative stress in different cell types
in our lab (Figures 1 and 2).
-CM-H2DCFDA (Invitrogen): this indicator is a chloromethyl derivative of H2DCFDA. CMH2DCFDA 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.
Figure 1. An oxidative burst was
detected by flow cytometry of cells
labeled with CM-H2DCFDA. Cells were
incubated with 120 nM CM-H2DCFDA. The
cells were washed and resuspended in PBS
(Control) or PBS with different concentrations
of H2O2. The samples were analyzed on a
LSRFortessa equipped with a 488 nm argon-ion
laser and a 530 ± 30 nm bandpass filter.
-GREEN DYE (Abcam): Reduced Glutathione (GSH) is important for maintaining redox
level of cells being involved in many cellular processes including the scavenging of free
radicals, drug detoxification, cell signaling, and cell proliferation. Non-fluorescent Green Dye
becomes strongly fluorescent upon reacting with thiol (including GSH in cells). In normal cells,
the Green Dye is accumulated primarily in cytosol, but it is partially translocated to
mitochondria in apoptotic cells while Green Dye staining intensity is decreased.
Figure 2. Mature erythrocytes stained
with Green Dye. Cells were prepared at a
density of 1 × 106 cells/mL on PBS. 200X Thiol
Green Dye was diluted 1/100 then 10L were
added of into 0,2mL of cell solution. Cells were
incubated at room temperature for 10-15
minutes. Re-suspend the cells in 1 mL of PBS or
PBS containing H202. After 5 min incubation,
the samples were analyzed on a LSRFortessa
equipped with a 488 nm argon-ion laser and a
530 ± 30 nm emission filter.