Seahorse

Real-time measurement of oxygen consumption rate (OCR) was performed using the Seahorse XFe96 FluxPak (102416-100, Agilent) and the Seahorse XFe Analyzer (Agilent). BMDMs and human monocytes were cultivated (and stimulated) in XFe96 microplates as described under Macrophage Activation and Stimulation. Before the analysis, cells were washed with unbuffered, DMEM-formulated Seahorse base medium (102363-100, Agilent), supplemented with 10 mM glucose (X997.2, Carl Roth) and 2 mM L-glutamine (25030-081, Gibco by Thermo Fisher Scientific) and incubated for 30 to 60 minutes at 37°C, without CO₂. For the analysis, cells were then loaded onto the Seahorse XFe Analyzer together with a precalibrated disposable cartridge. A mitochondrial stress test was performed. The assay was designed to repeat 5 cycles (of 3 min mixing followed by 3 min measuring) per phase. During the assay, 2 µM Oligomycin A (75351, Sigma by Merck), 1 µM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (C2920, Sigma by Merck), 1 µM rotenone (R8875, Sigma by Merck), and 1µM antimycin A (A8674, Sigma by Merck) were injected. After the assay, a nuclear staining with Hoechst (B2261, Sigma by Merck) was performed (10 µg/mL in cell culture medium for 45 minutes at 37°C). Cells were then washed with PBS and detached with 2 mM EDTA in PBS for 10 min on ice. Cell number was then determined on a BD LSRFortessa with high-throughput sampler (BD) using FACSDiva and FlowJo to normalize OCR to cell count per well. Human blood-derived monocytes were treated equally. However, as monocytes are nonadherent, cells needed to be centrifuged between washes (for 5 min at 300 g), and detaching was not necessary.