Lipophilic tracers

Pan PPAR-iMSC-EVs were incubated with 1 μg/mL DiR buffer for 10 min at 37 °C according to the protocol mentioned by Lipophilic Tracers (Invitrogen, Waltham, MA, USA). Next, the DiR-labeled pan PPAR-iMSC-EVs were centrifuged for 80 min at 100,000×g and 4 °C and washed with PBS (Gibco). Lastly, 200 or 400 μg of DiR-labeled pan PPAR-iMSC-EVs was resuspended in 0.1 mL of PBS and intravenously injected into C56BL/6 mice. At 24 h, DiR-labeled pan PPAR-iMSC-EVs were detected using an In Vivo Imaging System (Caliper Life Sciences, Waltham, MA, USA) at excitation and emission wavelengths of 740 and 790 nm, respectively. The intensity of the region of interest was plotted in units of the maximum number of photons per second per centimeter square per steradian (p/s/cm²/sr). The procedure for preparing DiD-labeled pan PPAR-iMSC-EVs was identical to the procedure described above. DiD-labeled pan PPAR-iMSC-EVs were used to treat the human primary hepatocytes or THP-1 macrophages for 24 h with or without each stimulus. At 24 h, DiD-labeled pan PPAR-iMSC-EVs were observed under a Nikon Eclipse Ti2-U fluorescent microscope (Nikon, Tokyo, Japan).

A stock solution of the lipophilic tracers (Invitrogen, Carlsbad, CA, USA) DiI (red fluorescence; Ex^{565 nm}, Em^{594 nm}) and DiO (green fluorescence; Ex^{484 nm}, Em^{501 nm}) were prepared in ethanol and DMSO. Exosomes isolated from culture medium were incubated with DiO and DiI (1 μM) dye for 30 min at 37°C. Exosomes were then washed with PBS and purified using ExoQuick™. Fluorescently labeled exosomes (20 μg/mL) were used to treat tumor cells (4T1), endothelial cells (SVEC), macrophages (Raw264.7), stem cells (mBs-MSC), fibroblasts (3T3), and dentritic cells (JAWS2). Fluorescent exosomes in cells were detected using a Zeiss LSM510 META confocal imaging system (Carl Zeiss, Thornwood, CA, USA). We also constructed a pCMV driven GFP/RFP-tagged CD9 vector and imaged exosomes with confocal microscopy.

IFN-γ-iMSC-EVs were incubated with 1 μg/mL DiR buffer for 10 min at 37 °C according to the protocol mentioned by Lipophilic Tracers (Invitrogen, Waltham, MA, USA). Next, the DiR-labeled IFN-γ-iMSC-EVs were centrifuged for 80 min at 100,000×g and 4 °C and washed with PBS (Gibco). Lastly, 100 μg of DiR-labeled IFN-γ-iMSC-EVs were resuspended in 0.2 mL of PBS and subcutaneously injected into DNCB-induced NC/Nga mice. After 8 h, DiR-labeled IFN-γ-iMSC-EVs were detected using an In Vivo Imaging System (Caliper Life Sciences, Waltham, MA, USA) at excitation and emission wavelengths of 740 and 790 nm, respectively. The intensity of the region of interest was plotted in units of the maximum number of photons per second per centimeter square per steradian (p/s/cm²/sr).

HA-iMSC-EVs were incubated with 1 μg/mL DiR or DiD buffer according to the protocol provided by Lipophilic Tracers (Invitrogen, Waltham, MA, USA) as described previously [34 (link)]. 500 μg of DiR-labeled HA-iMSC-EVs were resuspended in 0.05 mL of phosphate buffered saline (PBS) and intravenously or intramyocardially injected into rats. DiR-labeled HA-iMSC-EVs were detected at 6 and 24 h using an In Vivo Imaging System (IVIS, Perkin Elmer, Waltham, MA, USA). Human umbilical venous endothelial cells (HUVECs) or neonatal rat cardiac fibroblasts (NRCFs) were treated with DiD-labeled HA-iMSC-EVs. After 24 h, DiD-labeled HA-iMSC-EVs were observed under a Nikon Eclipse Ti2-U fluorescence microscope (Nikon, Tokyo, Japan).