Journal: Scientific Reports

Article Title: Therapeutic potential of human mesenchymal stromal cell-derived mitochondria in a rat model of surgical digestive fistula

doi: 10.1038/s41598-025-13887-3

Figure Lengend Snippet: Characterization of human MSCs-derived mitochondria. ( A ) TOM20 (green) staining and ( Bi – Biii ) MitoTracker red/TOM20 colabling of freshly isolated mitochondria indicating isolation of numerous viable mitochondria from hMSCs. ( C , D ) Scanned electron microscopy (SEM) and transmission electron microscopy using negative staining ( Di , Dii ) as well as cross-sectioned methods ( Ei , Eii ) depicting structurally intact mitochondria of various diameter ranging from 100–1200 nm. ( F , G ) Quality control analysis of isolated mitochondria by Western blotting of mitochondria membrane integrity markers and human OXPHOS complexes ( GI – GV ) showing co-expression of mitochondrial structural and functional proteins in mitochondria preparations (Mito) compared to their corresponding supernatant (SN) loaded with the same amount of protein (Uncropped western blots can be found in the Supplementary Fig. 1). ( H ) ILM of mitochondria samples using Videodrop particle counter showing the size distribution graph with a mean size of 366 nm and a representative interferometry image (depicting the particles in white circles appeared for a moment or the tracked particles shown in circles with orange color). IM inner membrane, OM outer membrane, IMS intermembrane space.

Article Snippet: Initially, isolated mitochondria were incubated either with an Alexa Fluor ® 488 Anti-TOMM20 (translocase of outer mitochondrial membrane 20 homolog) antibody [EPR15581-39] - Mitochondrial Marker (ab205486, abcam) or with a primary rabbit anti-mouse anti-TOM20 antibody (11802-1-AP, Proteintech) for 30 min followed by 30 min incubation in the dark with a secondary antibody (Goat anti-Rabbit IgG) conjugated with green fluorescent AlexaFluor 488 (A-11008, ThermoFisher).

Techniques: Derivative Assay, Staining, Isolation, Electron Microscopy, Transmission Assay, Negative Staining, Control, Western Blot, Membrane, Expressing, Functional Assay