Quantification of ROS production

Peritoneal macrophages were seeded in white or black 96-well plates for luminescence or fluorescence measurements, respectively. Macrophages were infected in ice-cold HBSS. To analyze extracellular ROS production, macrophages were incubated in 50 μM isoluminol (Sigma-Aldrich) and horseradish pereoxidase (3.2 U/ml) (Merck Millipore) in HBSS after infection. To analyze cytosolic ROS production, macrophages were incubated in 20 μM DCF (Thermo Fisher Scientific) in HBSS for 15 min at 37°C before infection. To analyze ROS production into the mitochondrial matrix, macrophages were incubated in 5 μM MitoSOX Red (Thermo Fisher Scientific) in HBSS for 15 min at 37°C before infection. Because Nox2-derived ROS oxidized MitoSOX Red already in the extracellular milieu, leading to false-positive MitoSOX Red fluorescence, Nox2^−/− macrophages were used for these experiments. After infection, plates were transferred on ice to the respective plate reader preheated to 37°C. Isoluminol luminescence or DCF fluorescence was measured at 1-min intervals using a TriStar² LB 942 Multimode Plate Reader (Berthold Technologies), and MitoSOX Red fluorescence was measured using a Tecan Infinite M 1000 microplate reader (Tecan Group).