3.2.9. DCFDA-Based Flow Cytometric Assay for Detection of Reactive Oxygen Species

In order to quantify intracellular ROS, we performed a flow cytometric assay based on the ROS-sensor 2′,7′-dichlorofluorescein diacetate (DCFDA) [52,53]. For acute effects of pentathiepins on intracellular ROS levels, the cells were stained with DCFDA first and subsequently incubated with 25 µM of compound for 10 min. For monitoring a treatment of 24 or 48 h, the cells were incubated with the respective pentathiepin (IC₉₀, see Table S4) and loaded with the DCFDA dye prior to analysis. Per condition, 250,000 cells were seeded in 2 mL of medium per well of a 6-well plate and allowed to adhere overnight. For measurement of acute ROS, the cells were stained with 1 mL of a 20 µM DCFDA-PBS solution for 30 min under standard incubation conditions. Afterwards, the solution was discarded, and the monolayer washed twice with 1 mL of PBS before 3 mL of medium containing 25 µM of pentathiepin was added for 10 min at 37 °C. After removing the solution and washing again with PBS, the cells were detached by using 0.5 mL of a diluted trypsin/EDTA-PBS solution (25%) and harvested with 1 mL of culture medium by carefully rinsing the wells. The cell suspension was transferred to 1.5 mL tubes, centrifuged for 5 min at 500× g, and washed with 1 mL of PBS. After removing the supernatant, each pellet was resuspended in 0.5 mL of PBS for measurement via the MACSquant Analyzer 10 at λ_Ex/_Em = 488/530 nm. Relative ROS levels resulting from treatment with pentathiepin were calculated by relating the fluorescence intensity of treated samples to the negative control. For the experiments covering periods of 24 and 48 h, the cells were incubated with the compounds first and stained with DCFDA afterwards. To assess the influence of additional GSH, we included 3 or 30 µM GSH to the medium containing 25 µM of pentathiepin, and the protocol was followed as described.