6 channel slides

ROS levels were measured using Cellular ROS/Superoxide Detection Assay Kit (Abcam, Cambridge, United Kingdom). Briefly, cells were seeded in 6-channel µ-Slides (Ibidi, Martinsried) at density 15,000 cells per well and treated with 50 μg Fe mL⁻¹ NPs for 24 h. Following this, cells were labeled with Oxidative Stress Detection Reagent (Green) for ROS detection and Superoxide Detection Reagent (Orange) according to the manufacturer’s instructions (Abcam, Cambridge, United Kingdom). Stained cells were imaged using spinning disk confocal microscopy IXplore SpinSR (Olympus, Tokyo, Japan). As positive control treatment with 1 mM H₂O₂ for 30 min was used.

Immunofluorescence staining was used in order to monitor NP subcellular localization, cytoskeleton dynamics and assessment of intracellular signaling events. Cells were seeded in 6-channel µ-Slides (Ibidi, Martinsried, Germany) at density 15,000 cells per well. Afterwards, cells were treated with different NP concentrations for indicated periods of time. After the treatment cells were washed with PBS and fixed with 4% paraformaldehyde in PBS pH 7.4 at room temperature for 10 min. Samples were permeabilized with 0.5% Triton X-100 before the staining. Immunofluorescence staining was performed on fixed cells using primary antibodies against different proteins summarized in Supplementary Table S2 and AlexaFluor 568- or AlexaFlour 488-conjugated secondary antibodies. Dilutions and catalogue numbers of used primary antibodies are given in Supplementary Table S2. Stained cells were imaged using spinning disk confocal microscopy IXplore SpinSR (Olympus, Tokyo, Japan). ImageJ software (NIH) was used for image processing.

Cells were seeded onto 96-well clear bottom plates (BD Biosciences, Prague, Czech) at a density of 5000 cells per well. After cells were incubated with cell culture media (EMEM, 10% FBS) containing different concentrations of nanoparticles for 24 h. For lysosomal stability assessment, we utilized an acridine orange (AO) assay. The AO assay was performed in accordance with our previously verified protocol [6 (link),34 (link)]. Briefly, cells with incorporated nanoparticles were labeled with 5 µg mL⁻¹ AO in culture medium for 15 min at 37 °C. Following nanoparticle treatment, cells were cultured at 37 °C for indicated periods of time and the intensity of orange fluorescence was then measured using a microplate reader SpectraFluor Plus (TECAN, Mannedorf, Switzerland). Readings were done in quadruplicates. Three independent experiments were performed for each measurement. Normalized fluorescence data are presented as means ± SEM.
Additionally, we evaluated the lysosomal integrity microscopically. Cells were seeded in 6-channel µ-Slides (Ibidi, Martinsried) at density 15,000 cells per well. Then cells were stimulated with NPs for 24 h. After, cells were labeled with 5 µg mL⁻¹ AO in culture medium for 15 min at 37 °C and imaged using spinning disk confocal microscopy IXplore SpinSR (Olympus, Tokyo, Japan). As positive control treatment with 20 % ethanol for 10 min was used.