Camel Leukocyte ROS Generation

ROS generation was performed in 96-well round-bottom microtiter plates (Corning, NY, USA) as described earlier [28 (link)] with modifications. Separated camel leukocytes (1 × 10⁶ / well) in RPMI medium were incubated with heat killed Staphylococcus aureus (50 bacteria/cell) for 20 min (37 °C, 5% CO₂). For the detection of ROS, dihydrorhodamine (DHR) 123 (Mobitec, Goettingen, Germany) was added to the cells (150 ng / ml final). To identify monocyte subsets, cells were labeled with monoclonal antibodies to CD14 and MHCII (see above). After washing, cells were analyzed by flow cytometry (FACSCalibur, Becton Dickinson Biosciences, San Jose, California, USA). The relative amount of generated ROS was determined by the mean green fluorescence intensity of gated monocyte subsets (based on CD14 and MHCII expression) after acquisition of 100,000 events (n = 15 animals).