Glomax multi reader

Human collagen IV protein (Abcam, Cambridge, MA, USA) was reconstituted in 100 mM sodium acetate and diluted (v:v, 1:1) in 100% molecular grade EtOH to facilitate drying. Matrices were dried in 96-well plates at 3 µg/cm² overnight at room temperature, washed in PBS and incubated with liposomes at 50 µM total lipid at 37 °C for 0–24 h. Unbound liposomes were rinsed 2× with PBS, and liposome binding was assayed in duplicate via the fluorometric detection of Rho-DPPE at 525 nm (GloMax Multi Reader, Promega, Madison, WI, USA). Static binding affinity was quantified as % lipid bound vs. 50 µM total lipid when compared to the fluorescence of an internal serial dilution curve of each relative liposome sample. Qualitative binding images were acquired by fluorescent microscopy using a Texas Red fluorescent filter at 400× under equivalent exposures across all groups (BX51 Olympus microscope, Olympus Q-color camera, Olympus Corporation, Shinjuku, Tokyo, Japan).

HeLa stably overexpressing Gluc-flag were seeded at a density of 5000 cells per well in 96-well assay plates. 24 h later the ferroptosis-modulating compounds (erastin, sorafenib, ferrostatin-1, GSH) were added in fresh medium and incubated for 24 h. Thereafter, medium was aspirated and replaced with medium containing BFA plus the respective ferroptosis-modulating compound, and the cells were grown for additional 2 h before the medium was exchanged with normal DMEM (without addition of any chemicals). 15 min after this medium exchange 50 µl of culture supernatant was transferred to a white, opaque 96-well plate. A volume of 20 µl freshly prepared Gaussia luciferase (Gluc) flash assay reagent (Pierce) was added and the luminescent signal was read after a 10 s integration time on a GloMax®-Multi reader (Promega).

Human cervix adenocarcinoma cells HeLa cells were obtained from ATCC-LGC Standards Repository (ATCC number CCL-2) and maintained in EMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS). To test the effect of guaianolides 1–3 on cell viability, cells were seeded in regular medium with 10% FBS (1 × 10⁴ cells/well in a 96-well multi-well system). After adhesion (approximately 3 h), medium was replaced with 0.4% FBS medium and cells were exposed to increasing concentrations (1–10–25–50–100 µM) of compounds for 24 h. After treatment, medium was removed and cells were fixed with a 10% trichloroacetic acid solution through a 1 h incubation at 4 °C. To remove fixative solution, cells were washed 3 times with distilled water and further incubated with a 0.4% SRB solution for 10 min at RT in the dark. Finally, cells were washed 3 times with a 1% acetic acid solution and let to air-dry overnight. The day after, to dissolve SRB-bound protein, 10 mM of Tris-HCl (pH 10.5) were added and absorbance (by means of optical density, OD) was measured by GloMaxmultireader (Promega) equipped with a 540 nm filter. OD values from vehicle-treated cells were considered as 100% of proliferation and results were expressed as percentage (%) of the control (vehicle alone). All compounds were dissolved in DMSO and the final percentage of solvent used was less than 0.3 % per well.