Dcf da

Intracellular ROS concentrations were measured by 2’,7’-dichlorofluorescindiacetate (DCF-DA) assay as previously described [5] (link). Briefly, J774 cells were plated at 1.5x10⁵ cells/well in a clear bottom and black-walled 96-well tissue culture plate overnight. The next day, the cells were washed and treated as indicated with LPS (1 μg/ml) in serum and phenol red free media for 3 h and with MitoQ (0.3–1.2 μM) for 2 additional hours. The media was then removed and replaced with fresh serum and phenol red free media containing 2.5 mM probenecid (Tocris Bioscience, Bristol, UK) with and without 10 μM DCF-DA (Molecular Probes, Eugene, OR) for 40 min at room temperature. The cells were subsequently washed in HBSS containing 2.5 mM probenecid for 5 min at 37 °C. The cells were then placed in serum-free and phenol red-free media with and without MitoQ (0.3–1.2 μM), read on a Synergy HTX Multi-Mode plate reader (BioTek) at 485_Ex/520_Em for 2 min, treated as indicated with ATP (1 mM) and read on the plate reader for an additional 30 min with one read per minute. The baseline read for each well and the average fluorescence of the group that was not treated with DCF-DA were subtracted from each subsequent reading and the areas under the curves were calculated using GraphPad Prism 7 for Windows (La Jolla, CA).