Simultaneous analysis of reactive oxygen species and reduced glutathione content in living cells by polychromatic flow cytometry.
Reactive oxygen species (ROS) are continuously produced in the cell as a consequence of aerobic metabolism, and are controlled by several antioxidant mechanisms. An accurate measurement of ROS is essential to evaluate the redox status of the cell, or the effects of molecules with the pro-oxidant or antioxidant activity. Here we report a cytofluorimetric technique for measuring simultaneously, at the single-cell level, hydrogen peroxide and superoxide anion, reduced glutathione (a main intracellular antioxidant) and cell viability. The staining is performed with the fluorescent dyes 2',7'-dichlorodihydrofluorescein diacetate (H2DCFH-DA), hydroethidine (HE), monobromobimane (MBB) and TO-PRO-3. This analysis is possible with new-generation flow cytometers equipped with several light sources (in our case, four lasers and an UV lamp), which excite different fluorochromes. This approach is extremely useful to study the balance between ROS content and antioxidants in cells receiving different stimuli, and to analyze the relationship between oxidative stress and cell death.
Cossarizza
Andrea
A
Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy. andrea.cossarizza@unimore.it
Ferraresi
Roberta
R
Troiano
Leonarda
L
Roat
Erika
E
Gibellini
Lara
L
Bertoncelli
Linda
L
Nasi
Milena
M
Pinti
Marcello
M
D002460:Cell Line; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism
D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D013481:Superoxides; D006861:Hydrogen Peroxide; D005978:Glutathione