Nitrite and TNF measurements

BMDM (2 x 10⁵ cells per well) in 24-well plates were infected with NMII at an MOI of 50. At 8 h post-infection, cells were mock treated or treated with 100 ng/ml LPS or 10 μg/ml zymosan (Sigma). For nitrite measurements, cells were also treated with 10 ng/ml murine TNF (PeproTech). At 24 h post-infection, culture supernatants were assayed for the relative level of nitric oxide by determining nitrite concentration with a Griess Reagent System (Promega). Culture supernatants stored at -20°C were assayed for TNF concentrations using a TNF ELISA kit (R & D Systems).