Opera phenix high content imaging instrument

hPSCs were seeded in 96-well plates (CellCarrier-96 ultra microplates, PerkinElmer, Waltham, MA, USA). At the start of the experiment, the differentiation of hPSCs was initiated using differentiation medium supplemented with 4 μM CHIR99021. C11-BODIPY (1 μM, Cat No. D-3861) and DRAQ7 (0.5 μM, Biostatus Cat No. DR71000) were added to each well 30 min before measurements. Also, ferroptosis inducer ML162 (0.5 μM, Aobious, Cat No. AOB1514) and FER-1 (500 nM) were included as control conditions in a subset of the wells in the experiment. Images were taken every 105 min for 17 h and a final measurement was performed at 24 h using the OPERA Phenix high content imaging instrument (PerkinElmer) with 20× water immersion objective. Using DRAQ7, dead cells could be identified from live cells. C11-BODIPY is used to detect lipid peroxidation events as oxidation of the probe results in a shift of the fluorescence emission peak from 590 nm to 510 nm when exited by 488 nm. This allowed the acquisition of the reduced and oxidized probe fluorescence in separate image channels. Visualization was done using the TIBCO Spotfire software package. Image analysis included single-cell segmentation and subsequent analysis of the percentage of dead cells (DRAQ7 positive cells) and/or calculations for the percentage of cells positive for lipid peroxidation (oxidized C11-BODIPY positive cells).

Live INS-1E cells were stained with the
DNA dye Hoechst 33342 (all cells), Caspase 3/7 activation dye CellEvent
Caspase-3/7 (apoptotic cells), and live cell impermeable DNA dye DRAQ7
(dead cells) all at 1:5000 dilution for 1.5 h. Cells were imaged at
the magnification 5× and 10× using an Opera Phenix High-Content
Imaging Instrument (PerkinElmer). Caspase-negative/positive and DRAQ7-negative/positive
cells were quantified using Harmony software (PerkinElmer).