Macrophage Cytotoxicity Assays.

J774 macrophage-like cells were grown to ∼60% confluency (∼1.5 × 10⁴ cells per well) in a 96-well microtiter dish. Bacteria were cultured in either ambient air or 5% CO₂ to an OD₆₀₀ of ∼1.0, and were then added to J774 cells at an MOI of 150. The plate was spun at 1,200 × g and then allowed to incubate in a tissue culture incubator for 3 h. Macrophage cytotoxicity was quantified by measuring lactate dehydrogenase release using Promega’s Cytotox96 Nonreactive Cytotoxicity Assay kit and a MultiskanEX plate reader (ThermoFisher Scientific) according to the manufacturer’s instructions.