Evaluating Intracellular ROS Levels

To evaluate intracellular ROS levels, dichlorodihydrofluorescein diacetate (DCFH-DA) assay was performed as previously described [22 (link)]. Cells were seeded in a 96-well plate at density of 10000 cells/cm², 4 replicates for each condition, treated with EGCG and SF for 72 hours soon after thawing, otherwise chronically during expansions. Cell culture medium was removed, and the 5 μM DCFH-DA was incubated in αMEM 1% FBS without phenol red 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 VICTOR multilabel plate reader (PerkinElmer).