2 7 dichlorofluorescein diacetate dcfda assay

To determine ROS production in the in vitro model, 2′–7′-dichlorofluorescein diacetate (DCFDA) assay (Abcam, UK) was used according to manufacturer’s protocols. Cells were cultured and as described and then washed and incubated with 10 µM DCFDA for 30 min at 37 °C at the dark. After washes with Phosphate Buffered Saline (PBS), cells were treated as described above in medium w/o phenol red. H₂O₂ 800 µM was utilized as a positive control. ROS production was immediately measured by detecting the fluorescent dichlorofluorescein (DCF), using Tecan Spark (Tecan, Swiss), at an Ex-485 and Em-535 nm. Measurements were performed every 30 min for six hours. Values reported were acquired by the ratio of fluorescence at a specific time point on fluorescence at time 0, which was determined after DCFDA incubation.

To examine apoptotic cell death, annexin V/propidium iodide (PI) (Thermo Fisher Scientific) staining was performed according to manufacturer’s protocol and cells were analysed by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA, USA). Annexin V and PI double-negative cells were considered viable, annexin V-positive and PI-negative (early apoptotic) cells and annexin V- and PI-positive (late apoptotic) cells were considered apoptotic, and PI only-positive cells were considered necrotic. To assess cellular ROS, a 2′,7′-dichlorofluorescein diacetate (DCFDA) assay (Abcam, Cambridge, UK) was performed according to the manufacturer’s protocol and analysed with the FACSCalibur. To assay autophagy activity, cells were labelled with anti-light chain 3 (LC3) (Abcam) followed by staining with Alexa488-conjugated goat anti-mouse IgG antibodies. Next, cells were analysed with FACSCalibur and data were analysed with the FlowJo software (ver. 7.6.5; Tree Star, Ashland, OR, USA).