Draq7

To evaluate cell viability, fresh cells were stained with DAPI (Molecular Probes) and DRAQ5 (Molecular Probes) and then assayed using a CyAn ADP High-Performance Flow Cytometer Analyser. To check for consistency, we tested other DNA dyes for dead/permeabilized cells: propidium iodide (PI; Thermo Fisher), DRAQ7 (Thermo Fisher) and 7-ADD (Thermo Fisher). The final dye concentrations used were 0.5 μg/mL, 0.05 μM, 1 μg/mL, 0.03 μM and 0.5 μg/mL, respectively.

Five thousand, 7500 or 10,000 cells per 96-well plate, 55,000 cells per 24-well plate or 300,000 cells per 6-well plate were seeded 24 h in advance, respectively. For KEAP1 siRNA knockdown, 20,000 cells were seeded in a 24-well plate on top of the transfection mix and incubated for 48 h followed by treatments for 24 h. Upon treatment, (Ferrostatin-1 [1 or 5 µM], RSL3 [0.1 µM or 1 µM], iFSP1 [10 µM], Erastin [0.37 µM], Sulfasalazine (SAS) [0.17 mM], Imidazole Ketone Erastin (IKE) [1.11 µM], ML210 [0.37 µM], ML162 [1.11 µM], TBHQ [25 nM]) cells were imaged using the 10× objective within the IncuCyte live cell imager (Sartorius). For dead cell quantification, 100 nM DRAQ7 (Thermofisher) were added to each well. For lipid ROS determination, cells were stained with 5 µM BODIPY C11. Cells were imaged for indicated timepoints every 2 h. Analysis for confluence, DRAQ7-positive (dead) or BODIPY C11-positive cells was performed using the Software IncuCyte 2021A (Sartorius).

All newly isolated PBMC were analysed before and after processing to determine baseline cell health characteristics and cell count. To determine viability, 10⁶ cells were incubated in RPMI with the viability stain DRAQ7 (ThermoFisher Scientific, UK) as per manufacturer's instructions and analysed using a MACSQuant MQ10 flow cytometer (Miltenyi Biotec, Ltd). Apoptosis was quantified by incubating 10⁶ cells in RPMI with CellEvent Caspase-3/7 Green Flow Cytometry reagent (Invitrogen, UK) at 37°C for 30 min. The PBMC were subsequently washed and stained with DRAQ7, as previously described, before analysis by flow cytometry. PBMC were gated to exclude doublets and debris, and the single-cell population was then analysed to quantify the dead (DRAQ7+) or apoptotic (CellEvent+) cells.

Cells were stained in HEPES buffer + 0.5% BSA (25–30 min, 4 °C), measured using a BD FACSCanto™ II flow cytometer (BD Biosciences), gated against specific isotypes, and analyzed using FlowJo™ (version 10.3; USA). Antibodies or reagents used were: (i) CD235a-PE (1:2500 dilution; OriGene cat#DM066R), CD49d-BV421 (1:100 dilution; BD-Biosciences cat#565,277), DRAQ7 (live/dead stain; 1:200 dilution; ThermoFischer Scientific cat#D15106); (ii) CD235a-PE (1:2500 dilution; OriGene cat#DM066R), CD71-APC (1:200 dilution; Miltenyi cat#130-d099-219); (iii) CD71-VioBlue (1:200 dilution; Miltenyi cat#130-101-631); (iv) DRAQ5 (nuclear stain; 1:2500 dilution; abcam cat#ab108410); (v) PI (live/dead stain; 1:2000 dilution; Invitrogen cat#P3566).

U2OS BAX/BAK DKO cells were seeded in a 48‐well plate and transfected with 50 ng pFRB‐EGFP‐BAX and 100 ng pFKBP‐EGFP‐DRP1 or 50 ng GFP‐BAX (or mutant versions of BAX) (Table 1) for 16 h. Cells were stained with DRAQ7 (Thermo Fischer Scientific) in a 1:3,000 dilution in DMEM to stain the DNA of dead cells. Dimerization of BAX and DRP1 was induced using 300 nM A/C Heterodimerizer (AP21967, Takara Bio) and apoptosis was induced using 1 µM ABT‐737 and 1 µM S63845 (Hölzel Biotech). Cell death and mitochondrial depolarization were measured using the IncuCyte bioimaging platform (Essen). Time‐lapse measurements of fluorescent intensity were performed every hour in nine positions of each well, which were averaged for analysis. Number of DRAQ7 positive cells was normalized to the number of transfected cells (based on GFP signal) using the cell‐by‐cell analysis mode. Data analysis was performed from n = 2 independent experiments each of them in technical duplicates. Levels of significance were determined using Student’s t‐test.

Primary human hepatocytes were seeded in 96-well black CellCarrier plates (PerkinElmer) at a density of 5 × 10³ cells per well. Following stimulations, cells were incubated 1 hr with 1 µg/ml Hoechst 33,342 (62249, Thermo Fisher Scientific) and DRAQ7 (D15106, Thermo Fisher Scientific) in serum-free basal medium. Each condition was imaged from triplicated wells and a minimum of 23 fields/well using Operetta high-content imaging system 1483 (PerkinElmer). Live and dead cells were quantified using Harmony v3.5.2 (PerkinElmer).

Embryos were incubated with a liberase-Blendzyme 3 (Roche) solution for 90 min at 33 °C, then dissociated and resuspended in 0.9× PBS-1% fetal calf serum, as previously described^{57 (link)}. We excluded dead cells by staining with SYTOX-red (Life Technologies), DAPI, or DRAQ7 (Thermo Fischer Scientific) stainings. Cell sorting was performed using an Aria II (BD Biosciences, software diva v6.1.3) or a BIORAD S3 cell sorter. For ROS detection, 25–30 embryos (kdrl:eGFP) were dissociated in 0.9% phosphate-buffered saline (PBS), 0.2 mg/mL liberase (Roche) for 2 h at 32 °C with agitation. Cell pellets were washed in PBS 0.9% containing 1% Fetal Calf Serum and centrifuged again for 5 mins. The supernatant was removed. Cells were placed in Hank’s Balanced Salt Solution (HBSS) with 5 mM CellROX deep red reagent (ThermoFisher) and incubated at room temperature for 30 min. Cell suspension was passed through a 40 mm filter prior to flow cytometry. Data were acquired on a LSR2Fortessa (BD Biosciences, software diva8.0.2) and analyzed with FlowJo.