Mitochondrial ROS quantification

Mitochondria isolation
Mitochondria were isolated from THP-1 cells or human monocytes using a sucrose gradient according to published methods (1). Briefly, cells were homogenized using a dounce homogenizer, and cell integrity was monitored via microscope after 25 strokes to ensure that 80-90% cell homogenization was obtained. Subsequently, cellular subfractions including endoplasmatic reticulum (ER), cytosolic (Cytopl.), crude (Mc) or pure mitochondria (Mp) were extracted from the same sample for immunoblotting for selected markers (2). Additionally, the Minute™ Plasma Membrane Protein Isolation and Cell Fractionation Kit (Invent Technologies #SM-005) was used to extract the plasma membrane (PM), cytosolic (Cytopl.) and other organelles (OO), following the manufacturer protocol. Tubulin as a cytosolic marker is absent in the mitochondria fraction.

Activation of purified mitochondria
The crude mitochondrial (Mc) fraction was further assessed for responsiveness toward C5a activation. Purified Mc was resuspended in mitochondria resuspension buffer prepared according to Wieckowski et al. (1). The solution containing Mc was distributed into equal volumes and treated with buffer or with increasing amounts of serum-purified C5a (5 to 100 ng/ml; CompTech) at 30°C with and without inhibitors for indicated time points. Mitochondria were then pelleted at 7000g, washed, and used in subsequent desired assays.

Mitochondrial ROS quantification
Crude mitochondria (Mc) were incubated with 1 µM CM-H2TMROS or 5µM MitoSOX (Thermo Fisher Scientific) reagent at 30°C for 30 min. Mc were washed with mitochondria resuspension buffer as described in (1), before treatment with PMX53 (10 µM) and activation with C5a (see above). Mc were plated in 96 Well White/Clear Bottom Plate (ThermoFisher, cat# 165306) that are optimized for fluorescence. The CM-H2TMROS or MitoSOX fluorescence was then measured using λex/em = 554/576 nm. The fluorescence intensity measured from mitochondria without CM-H2TMROS or MitoSOX treatment served as the basal background signal and was subtracted before analysis.

References
1. M. R. Wieckowski, C. Giorgi, M. Lebiedzinska, J. Duszynski, P. Pinton, Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells. Nat Protoc 4, 1582-1590 (2009).
2. Y. Suofu, W. Li, F. G. Jean-Alphonse, J. Jia, N. K. Khattar, J. Li, S. V. Baranov, D. Leronni, A. C. Mihalik, Y. He, E. Cecon, V. L. Wehbi, J. Kim, B. E. Heath, O. V. Baranova, X. Wang, M. J. Gable, E. S. Kretz, G. Di Benedetto, T. R. Lezon, L. M. Ferrando, T. M. Larkin, M. Sullivan, S. Yablonska, J. Wang, M. B. Minnigh, G. Guillaumet, F. Suzenet, R. M. Richardson, S. M. Poloyac, D. B. Stolz, R. Jockers, P. A. Witt-Enderby, D. L. Carlisle, J. P. Vilardaga, R. M. Friedlander, Dual role of mitochondria in producing melatonin and driving GPCR signaling to block cytochrome c release. Proc Natl Acad Sci U S A 114, E7997-e8006 (2017).