Mitosox

To determine the ROS levels released by cardiac mitochondria following their transfer to MSCs, cardiac mitochondria were isolated from RL14 cells previously stained with MitoSOX (5 μM, Invitrogen, Waltham, MA, USA, Cat#M36008) for 10 min. MSCs were then exposed to MitoSOX-labeled cardiac mitochondria for 24 h. MitoSOX fluorescence measurements were carried out with a Tecan Infinite M200 Pro plate reader combined with the acquisition software Magellan™ 7.2.
To scavenge ROS, cardiac mitochondria were treated with mitoTEMPO (500 µM, Sigma-Aldrich, Saint Louis, MO, USA, Cat#SML0737) in serum-free DMEM for 1 h prior to their exposure to MSCs.

BAECs were plated at density of 5 × 10⁴ for each well in 24-well plates. After incubation with PTH 0.1 nM for both 1 and 3 h, the cells were incubated with either 5 μM H₂DCFDA (Invitrogen) for 30 min at 37°C for total ROS (tROS) detection or with 5 μM Mitosox (Invitrogen) for 10 min at 37°C for mitochondrial ROS (mROS) detection, in a humidified atmosphere (5% CO₂, 95% air). After incubation, cells were washed twice with PBS, and fresh medium was added. The fluorescence was immediately measured by a plate reader (Tecan Infinite 200 Pro) using excitation/emission wavelengths of 492/520 nm for H₂DCFDA and 510/580 nm for Mitosox. Then, the cells were trypsinized and collected for cytofluorimetric analysis. In some experiments, cells were also treated with either 5 μM MitoTEMPO (Sigma-Aldrich), 100 nM calcipotriol hydrate, a vitamin D analogue (Sigma-Aldrich), or 10 μM Ru360 (Merck-Millipore). MitoTEMPO was administered 30 min before PTH, while both calcipotriol hydrate and Ru360 were coadministered with PTH.

Confluent ECs in 96-well plates were incubated with A-CRLPs for the times indicated in full growth medium without prior serum depletion. For incubations up to 1 h, cells were preloaded with dihydrorhodamine-1,2,3 (DHR; 10 μM) for 20 min before exposure to A-CRLPs. For longer incubations, ECs were challenged with A-CRLPs prior to addition of 10 μM DHR or 10 μM 2′7′-dichlorofluorescein-diacetate (DCF-DA) for 20 min, or 5 μM MitoSOX (ThermoFisher) for 10 min, as indicated. After washing in PBS, fluorescence was measured in a plate reader (Tecan, Switzerland) using excitation/emission wavelengths of 205/230 nm for DHR and DCF-DA, and 510/580 nm for MitoSOX.