Novocyte software

The LMP of the treated cells was measured using LysoTracker Green DND-26 (Thermo Fisher Scientific) staining. LysoTracker dye selectively accumulates in cellular compartments with low internal pH where it exhibits fluorescence. A decrease in this fluorescence is interpreted as LMP. In brief, the culture medium was aspirated after treatment and LysoTracker 50 nM in culture medium was added to the cells. After 15 min of incubation at 37°C, the cells were collected and the cell suspension was then transferred to a 5 ml FACS tube and analyzed on a flow cytometer within 10 min using Novocyte software (ACEA Biosciences Inc.).

Lipid peroxidation was determined by flow cytometry with C11-BODIPY 581/591 (D-3861). Oxidation of a component of the C11-BODIPY fluorophore shifts the fluorescence emission from red to green, and a change in the ratio of green to red fluorescence was used as an indicator of an increase in lipid peroxidation. The samples were collected and stained with 1 μM C11-BODIPY and then incubated in the dark in a water bath at 37°C for 15 min. The cell suspension was then transferred to a 5 ml FACS tube and analyzed on a flow cytometer within 10 min using Novocyte software (ACEA Biosciences Inc.).

ROS generation was determined by flow cytometry with dihydroethidium (DHE D-1168). DHE is oxidized by ROS into 2-hydroxyethidium (2-HE) (emission at 605 nm) and fluoresces red. The samples were collected and stained with 5 μM DHE and then incubated in the dark in a water bath at 37°C for 15 min. The cell suspension was then transferred to a 5 ml FACS tube and analyzed on a flow cytometer within 10 min using Novocyte software (ACEA Biosciences Inc.).

After 48 h of treatment, PBMCs were centrifuged, and the supernatants were stored at -20° C for the assessment of cytokine release. Cell pellets were washed with PBS, suspended in 200 µl of PBS, and stained at 4° C in the dark for 30 min with specific PE-conjugated antibody against human CD54 or FITC-conjugated antibody against human CD86 or with isotype control antibodies, following supplier’s instructions. All the antibodies were purchased from BD Biosciences (Franklin Lakes, New Jersey, US). After incubation, cells were centrifuged and suspended in 500 µL of PBS. The % of positive cells was analyzed using Novocyte 3000 flow cytometer (Acea Bioscience Inc., Agilent Technologies, Santa Clara, California, US) and data were quantified using Novocyte software (Acea Bioscience Inc.). 10′000 viable cells were analyzed for % of positivity to the respective marker. The % of isotype control was subtracted from the % of CD86/CD54 stained cells. Changes in CD86/CD54 expression are reported as SI calculated on DMSO-treated PBMC (vehicle control) set at 1. The gating strategy is reported in Supplementary Fig. 1 and representative dot plots are reported in Supplementary Fig. 2.