Measurement of reactive oxygen species (ROS) by flow cytometry

The 2′,7′-Dichlorodihydrofluorescein diacetate (H₂DCFH-DA) (Sigma-Aldrich, Germany) is a probe used for the highly sensitive and quantifiable detection of ROS. The non-fluorescent H₂DCFH-DA diffuses into the cells and is cleaved by cytoplasmic esterases into 2′,7′-dichlorodihydrofluorescein (H₂DCF) which is unable to diffuse back out of the cells. In the presence of hydrogen peroxide, H₂DCF is oxidized to the fluorescent molecule dichlorofluorescein (DCF) by peroxidases. The fluorescent signal emanating from DCF can be measured and quantified by flow cytometry, thus providing an indication of intracellular ROS concentration (Kuete et al. 2011c; Bass et al. 1983; Cossarizza et al. 2009). Briefly, 2 × 10⁶ CCRF–CEM cells were resuspended in PBS and incubated with 2 µM H₂DCFH-DA for 20 min in the dark. Subsequently, cells were washed with PBS and resuspended in RPMI 1640 culture medium containing different concentrations of PEF, AML, AMS or DMSO (solvent control), or hydrogen peroxide (H₂O₂; positive control). After 24 h of incubation, cells were washed and suspended in PBS. Subsequently cells were measured in a FACSCalibur flow cytometer (Becton–Dickinson, Germany). For each sample 1 × 10⁴ cells were counted. DCF was measured at 488 nm excitation (25mW) and detected using a 530/30 nm bandpass filter. All parameters were plotted on a logarithmic scale. Cytographs were analyzed using FlowJo software (Celeza, Switzerland). All experiments were performed in triplicate.