To evaluate intracellular ROS levels, dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma Aldrich, St Louis, MO, USA) assay was performed similarly to as previously described [51 (link)]. After cell treatments, cell culture medium was removed, and the 5 μM DCFH-DA was incubated in PBS for 30 min, at 37 °C and 5% CO2. The cell culture plate was washed with PBS, and fluorescence of the cells was read at 485 nm (excitation) and 535 nm (emission) using the multiwall reader Appliskan (Thermo-Fisher Scientific, Vantaa, Finland). Cellular autofluorescence was subtracted as a background using the values of the wells not incubated with the probe.
Similarly, to evaluate reduced GSH levels, monochlorobimane (MCB, Sigma Aldrich, St Louis, MO, USA) assay was performed as previously reported [52 (link)]. Cell culture medium was removed, and 50 μM MCB was incubated in PBS for 30 min, at 37 °C and 5% CO2. The cells were washed in PBS, and their fluorescence was measured at 355 nm (excitation) and 460 nm (emission).
Similarly, to evaluate reduced GSH levels, monochlorobimane (MCB, Sigma Aldrich, St Louis, MO, USA) assay was performed as previously reported [52 (link)]. Cell culture medium was removed, and 50 μM MCB was incubated in PBS for 30 min, at 37 °C and 5% CO2. The cells were washed in PBS, and their fluorescence was measured at 355 nm (excitation) and 460 nm (emission).
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