Eight well chambers

Stable FLIM cells were generated using lentivirus that expresses PGK-H2B-GFP and PGK-H2B-mCherry, and FACS sorted to produce populations with homogenous expression. For FLIM-FRET experiments, cells were seeded into eight-well chambers (ibidi) and treated with DMSO, 750 nM A23817, or 0.4 U/ml Thrombin for 15 min, before being fixed in 4% PFA for 10 min. Lifetime measurements were taken on a Leica TCS SP8 system, using a white light laser with a repetition rate of 80 MHz and an excitation wavelength of 488 nm. H2B-GFP emission was detected over an emission range of 495–530 nm. Data were fitted using the FLIMfit software tool developed at Imperial College London.

The mesothelial clearance assays were performed based on a protocol previously described [44 (link)] with minor alterations. MeT5A cells stably expressing EGFP protein were seeded in eight-well chambers (Ibidi) coated with 10 µg/mL of collagen type I (Millipore, Burlington, MA, USA) and 5 µg/mL of fibronectin (Sigma) and incubated for 24 h to form confluent monolayers. In parallel, aggregates of ovarian cancer cells were generated by seeding 350 cells per well in 96-well plates with a round bottom preciously coated with polyHEMA. After incubating for 24 h, the OVCA aggregates were transferred to the wells containing the mesothelial monolayers. Using the inverted motorized epifluorescence microscope Leica DMI 6000-time lapse, live cell imaging was performed for 24 h. To quantify the mesothelial clearance area, the non-fluorescent surface created by the invading ovarian cancer aggregate in the EGFP mesothelial monolayer was measured at 18 h of co-culture and divided by the initial two-dimensional area of the aggregate at the initial seeding time. All measurements were taken using the Fiji software. More than eight multicellular aggregates were imaged by each cell line in each experiment in a total of three biological experiments.

Cells were seeded in DMEM in IBIDI eight-well chambers (Ibidi, Germany) 24 h before the experiment. The day after, cells were washed with PBS to replace the media by phenol red-free DMEM (Sigma-Aldrich, Germany) supplemented with FBS and antibiotics. Cells were loaded with 1-µM Fluo-4 AM, 2-µg/mL PI, or 1 µM of C11 BODIPY 581/591 for 30 min at 37 °C. All images were acquired with a Zeiss LSM 710 ConfoCor3 microscope (Carl Zeiss, Jena, Germany) or a gSTED Leica confocal microscopy equipped with incubator at 37 °C and 5% CO₂. Time-lapse imaging with z-stack acquisition was carried out before and after ferroptosis induction. Transmitted light and fluorescence images were acquired through a Zeiss C-Apochromat 40X, NA = 1.2 water immersion objective or a 63X, NA = 1.2 oil immersion objective onto the sample. Excitation light came from Argon ion (488 nm) or HeNe (561 nm) lasers. All images were processed in Fiji. The percentage of Fluo-4 AM-positive cells was calculated at each time point. For this, individual cells were automatically detected based on the fluorescence of the Fluo-4 AM. To define cells as positive, we arbitrarily set a fluorescence threshold in which the percentage of Fluo-4 AM-positive cells did not exceed 5% in the negative control. The total number of cells in each condition was determined in the transmitted light images.