Mitotracker green

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).

To mimic ICH in PC12 cells, OH was added to the PC12 cell culture medium at a concentration of 10 μM for 12 hours. CsA was added (5 μM) at the same time. After 12 hours, the function of mitochondria in the PC12 cells in each group (Figure 1A) was examined as described in a previous study (Sarkar et al., 2020). Mitochondrial membrane potential (ΔΨm) were quantified in live cells using confocal microscopy. Mitochondria were labeled with MitoTracker®Green (Cat# 9074, Cell Signaling Technology, Boston, MA, USA), and nuclei were stained using Hoechst 33342 (Cat# 14533, Sigma, St. Louis, MO, USA). We used 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1, 0.5 μg/mL, Beyotime, Shanghai, China, Cat# C2006) and tetramethylrhodamine methyl ester (TMRM; 30 nM; Cat# T668, Invitrogen, Carlsbad, CA, USA) to measure ΔΨm. JC-1poly emits red fluorescence and labels mitochondria with high ΔΨm, and JC-1mono emits green fluorescence and labels mitochondria with low ΔΨm. The changes in ΔΨm are expressed as changes in the JC-1poly (red)/JC-1mono (green) fluorescence ratio. A decreased TMRM signal indicates a low ΔΨm.

Adult primary cardiomyocytes were isolated and cultured on costar 6 well plate. Then the mitochondria isolated from mouse heart were stained with Mitotracker green (Cell Signaling, Danvers, MA. USA) and incubated with cardiomyocytes after interfering with 2 μM doxorubicin. The cellular death and mitochondrial internalization were detected by living cell imaging analysis system (BioTek, Winooski, VT, USA).