Mitochondrial Function in LPS-Tolerant Macrophages

LPS-tolerance might be associated with cell-energy adaptation. Therefore, several parameters of mitochondria were explored. As such, 200 nM of Mitotracker Red CMxROS (Molecular probe) was added to each well and incubated at 37°C for 15 min before removal. Then, cells were fixed with cold methanol at −20°C for 15 min, washed twice with PBS, and photographed by an IX81 inverted microscope (Olympus, Tokyo, Japan). Energy metabolism profiles with estimation of glycolysis were performed and assessments of mitochondrial oxidative phosphorylation with extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were carried out by Seahorse XFp Analyzers (Agilent, Santa Clara, CA, USA) on macrophages at 1 × 10⁵ cells/ well by Seahorse Wave 2.6 software as previously described (21 (link)).