Seahorse XF Analyzer Respiratory Assay

Cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were detected using XF Cell Mito Stress Test (Agilent) measured by the extracellular flux analyzer XFe96 (Seahorse Bioscience, Houston, TX, United States) in the Hyp-ACB Platform in Sapienza University. Microglia were cultured on XFe culture miniplates (coated with poly-L-Lys) for a total of 4 days (40 × 10³ cells/well). After 2 days microglia were stimulated with 100 or 10 ng/ml of LPS for 24 h and 100 nM fractalkine for further 24 h. Both treatments show the same trend but only the change observed with 10 ng/ml was statistically significant and therefore only these data were shown. It is likely that 100 ng/ml treatment leads to a dramatic OCR reduction and in such a contest the variation due to CX3CL1 treatment are below the sensibility of the system. The sensor cartridge for XFe analyzer was hydrated in a 37°C non-CO₂ incubator a day before the experiment. According to the manufacturer instructions, stressors concentrations were optimized and added as follows: 1 μM oligomycin as complex V inhibitor, 1 μM FCCP (uncoupler agent) and 0.5 μM rotenone/antimycin A (inhibitors of complex I and III). During sensor calibration, cells were incubated 1 h in a 37°C non-CO₂ incubator in 180 μl assay medium (XF base medium supplemented with 10 mM glucose, 10 mM pyruvate and 2 mM L-glutamine at pH 7.4, was used to wash the cells and replace the growth medium). OCR was normalized for total protein/well/40 × 10³ cells. Each sample/treatment was analyzed in at least 8 wells for experiment; the figure represents one sample experiment. Two independent experiments were carried out on two different primary microglial culture preparations.