Lasx 3d software

Cells were stained with a 2.5 µM MitoSOX probe (M36008, Thermo Fisher Scientific, Waltham, MA, USA) or a 200 nM TMRE probe (T669, Thermo Fisher Scientific) for 20 min at 37 °C and examined using a confocal microscope (TCS SP8; Leica, Wetzlar, Germany). Hoechst (62249, Thermo Fisher Scientific) was used to visualize the nuclei, as previously described [30 (link)]. 3D reconstruction was assessed using Leica LasX 3D software. For TOM-20 immunofluorescence staining, cells were analyzed according to manufacturer’s instructions. To evaluate mitochondrial fragmentation, approximately 100 mitochondria in three randomly chosen fields were selected, and their size/shape was measured with ImageJ software (https://imagej.net/imaging/particle-analysis) (accessed on 9 January 2020). The percentage of mitochondria smaller than 0.6 μm² was reported as the Circularity Factor.

The bone thick sections were imaged with a confocal microscope Leica TCS SP8 (Leica Microsystems, Germany) equipped with a pulsed white light laser (470–670 nm), Acousto-Optical Beam Splitter (AOBS), and two internal hybrid single photon counting detectors, which was operated by Leica Application Suite X program (Leica Microsystems, Wetzlar, Germany). Z-stacks of 184.52 μm (x) × 184.52 μm (y) × 50 μm (z) with an X/Y resolution of 1024 × 1024 pixels were obtained by the sequential overlapping of 100 continuous images at an increment of Z-axis optical section of 0.5 μm. Two scans were performed along the XY plane, sequential in the Y axis, to obtain an image reconstruction of a complete column; see Supplementary Videos S1–6. The Leica LAS X 3D software was used for the 3D projection and the IMARIS v. 7.1.1. software (Bitplane, Switzerland) to obtain the cell structural parameters of the chondrocytes: cell volume, integrated optical density (IOD), and sphericity. The sequence of structural variation along a complete column was analyzed by measuring a total of 300 chondrocytes for each experimental group.