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3 protocols using ML162

The following compounds were used: polybrene, puromycin, buthionine sulfoxamine, paraquat, L-ascorbic acid (Sigma-Aldrich), blasticidin (Invivogen), RSL3 (Selleckchem), ML162 (Aobious), erastin (Tocris), ML21051 (link) (Selleckchem), dithiothreitol (Biovision), ferrostatin-1 (Tocris), and matrigel (Corning), Egg PC (Sigma), STY-BODIPY35 (link), DTUN32 (link), α-tocopherol (Sigma, purified by flash chromatography before use), CoenzymeQ10 (Alfa Aesar), tetrahydrobiopterin (BH4) and dihydrobiopterin (BH2) (Cayman Chemicals), NADPH (Alfa Aesar), dihydrofolate reductase (R&D Systems), methotrexate (US Biological), superoxide dismutase (Sigma), catalase (Sigma). All purchased compounds had a reported purity value ≥ 98%. QM385 was kindly provided by Dr. Clifford Woolf.
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Spheroids were collected at day 1 and 10 (L929) and day 1 (SKOV) and seeded at a density of 80 spheroids per well in a 96-well plate in duplicate. A 2D control was seeded as well at a density of 10,000 cells per well. Cells were stained either immediately after seeding (spheroids) or after 24 h (2D culture) using the optimal Sytox Green (L929) or Sytox Blue (SKOV) concentration. An apoptosis inhibitor (zVAD-fmk, BACHEM, Budendorf, Switzerland), necroptosis inhibitor (Nec-1s, Abcam, Cambridge, UK) and multiple ferroptosis inhibitors (Fer-1, DFO and α-Toc, Sigma, Saint Louis, MO, USA) were added 30 min before cell death induction with 5 µM ML-162 (AOBIOUS, Gloucester, MA, USA), an inhibitor of GPX4 and thus an inducer of ferroptosis. After 24 h, Sytox intensity was measured using the Tecan Spark microplate fluorescence reader. Afterwards, cells were permeabilised with the optimal Triton X-100 concentration (found in the previous experiment) to obtain 100% of cell death. After 2 h of incubation, Sytox intensity was measured again.
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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).
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