Cellsens dimension 2.3 imaging software

The Intracellular Hydrogen Peroxidase assay kit (Sigma-Aldrich, Taufkirchen, Germany; MAK164) was used for imaging of H₂O₂ production. A549 cells (1.5 × 10⁴) were seeded into a black/clear-bottom 96-well plate and incubated for up to 24 h, followed by 4 h serum starvation. Cells were then incubated with 100 µL of the Fluorescent Peroxide Sensor (FITC) for 15 min and washed with PBS. To stain the nuclei, 1 drop of NucBlue™ Live ReadyProbes™ Reagent (Thermo Fisher, Hoechst 33342) was added to cells in PBS and they were visualized using an Olympus iX70 fluorescence microscope and the Olympus cellSens Dimension 2.3 imaging software.

A total of 1 × 10⁴ or 2 × 10⁵ A549 cells were seeded in 4-well chamber slides or on coverslips in 6-well plates and incubated for up to 24 h at 37 °C. Cells were serum starved for 2–4 h followed by MPO incubation for the indicated timepoints. To investigate MPO uptake by A549 cells, the cells were incubated with heparin (150 µg/mL) for 1 h before adding MPO (10 µg/mL) for 2 h. Cells treated with ddH₂O or DMSO (0.2%) were used as the controls. Cells were rinsed with PBS (3 × 2 min) before fixing and permeabilizing with ice-cold methanol for 20 min at −20 °C. Cells were washed with PBS (3 × 2 min on shaker) and incubated with blocking solution (5% goat serum + 5% BSA in PBS with 0.1% TritonX) for 60 min at room temperature (on shaker). The primary antibody (rabbit anti-MPO 1:500; Cell Signalling, Danvers, MA, USA) was prepared in an antibody diluent (1:10 blocking solution in PBS-TritonX) and incubated overnight at 4 °C. After washing the cells with PBS (3 × 2 min on shaker), they were incubated with the secondary antibody (goat anti-rabbit AF488, 1:500) for 1 h at room temperature in the dark. After washing the cells with PBS (5 × 2 min on shaker), they were mounted using VECTASHIELD with DAPI. The cells were visualized using an Olympus iX70 fluorescence microscope and the Olympus cellSens Dimension 2.3 imaging software with Z-stacking to allow imaging of different cell layers.