Cytotoxicity assay

Peritoneal macrophages were collected as described above. After 1 hour of adhesion, macrophages were incubated with different concentrations of 2HF (0 μM- 96 μM) without infection for 72 hours (37°C and 5% of CO₂). The macrophage viability was accessed using resazurin (20% v/v), which was reduced to resorufin after contacting viable cells, and the fluorescence (ex/em: 560/590 nm) was measured by a SpectraMax M2—Molecular Devices, Silicon Valley, USA. The cytotoxicity concentration (CC₅₀) was determined by logarithmic regression analysis using GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA). The experiments were performed thrice.