Lionheart fx livingcell imaging analysis system

For in vitro imaging,
MitoTracker Green and Red (Cell Signaling Technology, Danvers, MA,
USA) were used to incubate with isolated mitochondria and receptor
cells, respectively. The combination of Cy5 marked PEP–TPP
and MitoTracker Green-labeled mitochondria was imaged by a confocal
microscope (Thermo Fisher Scientific). To observe the dynamic internalization
of transplanted mitochondria, PEP(Cy5)–TPP–mitochondria
was added into the culture medium. Then the fluorescence intensity
of MitoTracker Green and Cy5 was detected by the Lionheart FX living
cell imaging analysis system (BioTek, Winooski, VT, USA).
For
in vivo imaging, to detect the retention of transplanted PEP(Cy5)–TPP–mitochondria
(GFP), mouse hearts were imaged by a fluorescence detection system
(IVIS Lumina XRMS, USA) after reperfusion for 3, 6, and 24 h. Besides,
the Cox4i1-GFP marked mitochondria of formalin-fixed heart tissue
sections were imaged with a fluorescence microscope (Thermo Fisher
Scientific).

MitoTracker red and green (Cell Signaling Technology, Danvers, MA, USA) were used to label the mitochondria of recipient and donor cells, respectively. To determine the ability and dynamic internalization of transplanted mitochondria, MitoTracker green-labeled mitochondria were isolated and then co-cultured with recipient cells. For recipient H9c2 cells, the fluorescence intensity was detected by flow cytometry (BD Biosciences, San Jose, CA, USA) at different time points (0 h, 1 h, 3 h, 6 h, 12 h). For cardiomyocytes, the fluorescence intensity was examined by the Lionheart FX living cell imaging analysis system (BioTek, Winooski, VT, USA). Localization of green fluorescence-labeled mitochondria from donor cells into recipient cells with red fluorescence-labeled mitochondria was imaged by a confocal microscope (Thermo Fisher Scientific) after co-culture for 3 h.