Lysosomal Integrity Evaluation of Nanoparticles

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.