Mitosox red

Cells were seeded on 35-mm glass bottom dishes (MatTek Corporation, Ashland, MA) and incubated with the superoxide indicator MitoSOX™ Red (5 μM, Invitrogen, Eugene, OR) for 15 min at 37 °C. Cells were washed with PBS, the media was replaced with exposure media, and the dish was inserted into a closed, thermo-controlled (37 °C) stage top incubator (Tokai Hit Co., Shizuoka-ken, Japan) atop the motorized stage of an inverted Nikon TiE fluorescent microscope (Nikon Inc., Melville, NY) equipped with a 60X oil immersion optic (Nikon, CFI PlanFluor, NA 1.43) and NIS Elements Software. MitoSOX™ Red was excited using a Lumencor diode-pumped light engine (SpectraX, Lumencor Inc., Beaverton OR) and detected using a DsRed longpass filter set (Chroma Technology Corp) and ORCA-Flash4.0 sCMOS camera (HAMAMATSU Corporation, Bridgewater, NJ). Data was collected on approximately 80 to 100 cells per stage position, with eight to ten stage positions in each of the separate experiments for 180 min. Data were analyzed using NIS Elements (Nikon Inc., Melville, NY). Data from three independent analyses for each particle size were used in the statistical calculations. Stage positions in which the particles did not result in alveolar macrophage (AM) generation of ROS were not used in the statistical analysis.