Quantification of Cellular Redox Status

A total of 24 h post treatments, we washed the spheroids twice with PBS and incubated them in 50 µL TrypLE express reagent (12604-021, Life Technologies Europe B.V., Bleiswijk, The Netherlands) for 15 min at 37 °C in a 96-well plate. Immediately after, we added fresh warm culture medium and we dissociated the spheroids with gentle mechanical force using a Pasteur pipette. After the cells were centrifuged at 1000 rpm up to 3 min, we measured the glutathione (GSH) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels. We quantified the total GSH and genomic DNA in 10⁴ cells from dissociated spheroids. We measured the GSH level using a luminescence-based assay (Promega, Madison, WI, USA) following a standard molecular biology protocol [37 (link),38 (link)]. We used the genomic DNA extracted from the same number of tumor cells for the detection of the 8-OHdG level using an oxidative DNA damage ELISA kit (Cell Biolabs, inc. San Diego, CA, USA).