Pkh26 fluorescent dye

Exosomes were labeled with pyridoxal kinase 26 (PKH26) fluorescent dye (Sigma-Aldrich, St. Louis, MO, USA): 4 μL of PKH26 fluorescent dye was supplemented to 1 mL Dilument C (Beyotime, Shanghai, China) solution to prepare 4 µM PKH26 dye, which was then added with 200 μg of exosomes. The above two solutions were subsequently mixed gently for five min and then centrifuged for 2 h at 100,000×g and 4 °C for 2 h, resuspended, and centrifuged again as before prior to the collection of the fluorescent-labeled exosomes.

Mesenchymal stem cells (MSC, Lonza, Inc., Switzerland) were cultured in MSC growth media supplemented with MSC growth supplement, L-glutamine, and Gentamicin-Amphotericin-B (Lonza, Inc., Switzerland) as previously reported [28 (link)]. Cultured MSC (60–70% confluence) were harvested between passages 5–8 using a standard procedure with 0.25% trypsin-EDTA (Gibco-Thermo Fischer, USA) [29 (link)]. Prior to injection, MSC were labeled using PKH26 fluorescent dye (Sigma-Aldrich, UK) according to the manufacturer’s instructions and as reported previously [28 (link)]. The efficacy of MSC labeling with PKH26 fluorescent dye was confirmed prior to cell delivery by flow cytometry and confocal microscopy. Briefly, unstained MSC controls and PKH26 labeled MSC were fixed in 4% paraformaldehyde and analyzed using a Fortessa flow cytometer (Becton Dickinson, USA). For confocal microscopy, cells were spun onto positively charged lysine-coated slides and counterstained with DAPI (Vector Laboratories, USA). Next, slides were examined using Zeiss Meta confocal microscope and analyzed with ZEN software (Zeiss, Germany). The viability and number of hMSC before and after PKH26 labeling were tested using 0.4% Trypan Blue.

hUC-MSCs were cultured with 1 ng/mL IL-6 (Sigma-Aldrich) in the medium for 48 h, and the supernatant was collected for exosome purification using an ExoQuick ULTRA EV isolation kit (SBI, Palo Alto, CA, USA). The characterization of exosomes was confirmed by electron microscopy and particle size by NanoSight analysis (Shanghai Oe Biotech Co., Ltd.) as described [19 (link)]. The expression of the exosome-specific marker TSG101 (SBI) and the EV-related protein markers CD63 and CD81 (SBI) were analyzed by western blotting. miRNAs were extracted from exosomes using TRIzol reagent (Invitrogen, USA) for qPCR analysis or sequencing at Shanghai Oe Biotech. To identify the transport of exosomes, exosomes were labeled with PKH26 fluorescent dye (Sigma-Aldrich) [19 (link)].

Tumor cells (HepG2, A549, MGC-803 and primary HCC cells) at the logarithmic growth stage were collected, inactivated by 30 GyX irradiation, and mixed and cultured with PKH26 fluorescent dye (Sigma-Aldrich, USA). DCs cultured with CFSE fluorescent dye (Sigma-Aldrich, USA) were blended with tumor cells at 2:1 ratio, and the mixture was added into a 50 ml centrifuge tube. During this process, PEG (Sigma, USA) was slowly and gently added along the wall of the tube and was water-bathed for 5 min at 37 °C. The diluted collagen I (Sigma, USA) was then added and soaked in a 37 °C water bath for 30 min.
The cells, upon PBS washing, were suspended again. The fusion of tumor cells and DCs was viewed by fluorescence microscopy (Nikon ECLIPSE 80i, Nikon, Japan). To assess the DC maturation, the fusion cells were collected after 7 days of culture, and MHC II(Abcam, UK, Cat No. ab55152), CD80(Abcam, UK, Cat No. ab134120), and CD86(Abcam, UK, Cat No. ab239075) expression levels were tested by flow cytometry (Backman CytoFlex S). FlowJo v10.0 was utilized for data analysis. The fluorescence intensity of fusion cells was captured under a fluorescence microscope.

To identify the internalization of hUC-MSC-sEVs into macrophages, hUC-MSC-sEVs were stained with PKH26 fluorescent dye (Sigma-Aldrich, USA) according to the manufacturer’s instructions and centrifuged again to remove contaminating dye. Then, the PKH26-labeled hUC-MSC-sEVs (10 μg/ml) were co-cultured with PMA-treated macrophages. After 6h, the macrophages were washed with PBS and fixed in 4% paraformaldehyde for 15min. Thereafter, the nucleus was stained with DAPI (Solarbio, China). Finally, the cells were observed and photographed with the laser scanning confocal microscope (LSM800, Zeiss, Germany).

Exosomes from POL cells were labeled with PKH26 fluorescent dye (Sigma) according to the manufacturer’s instructions. For POL cells transfected with miR-N.C. mimics or miR-199a-1-5p mimics, the mimics were labeled with Cy5 fluorescent dye (Sigma) according to the manufacturer’s instructions. Dye-labeled exosomes was added to OL cells and incubated at 37 °C for 18 h. The nuclei were stained with DAPI for 15 min and washed twice with PBS before confocal microscopy (TCS SP2, LEICA) analysis. A time course (0, 6, 12, 18 h) of exosomes uptake was conducted.