Pkh67 membrane dye

Purified exosomes were injected into the mouse retro-orbital venous sinus in a total volume of 100 μL PBS. For in vivo education experiments, mice received 5 μg of exosomes every other day for 21 days. Retro-orbital injection of PBS was used in control groups. For in vitro education, exosomes (10 μg/mL) were added into the culture medium (CM) of ImKC for 3, 6, 12, 24 h. For exosome-tracking experiments, exosomes were labeled using PKH67 membrane dye (Sigma, Shanghai, China), followed by 100,000×g ultracentrifugation for 70 min, and labeled exosomes were resuspended in PBS. In experiments involving the evaluation of exosome incorporation, labeled exosomes were injected retro-orbitally into the mice or added into the CM of ImKC 24 h before immunofluorescence analysis for exosome cells.

RCC cells were cultured in medium with 10% exosome-free medium (System Biosciences) for 72 h before the isolation of exosomes. The supernatant was collected and centrifuged at 300 × g for 10 min, 2000 × g for 10 min, 10,000 × g for 30 min, in turn. After filtration with a 0.22-pm filter (Millipore, USA), the filtrate was centrifuged at 120,000 × g for 70 min twice (Beckman Coulter, USA). The pellets were resuspended with PBS and prepared for the subsequent experiments. For transmission electron microscopy, exosomes were fixed with 2% paraformaldehyde and placed on 200-mesh Formvar-coated grids. The grids were then stained using 2% phosphotungstic acid for 2 min and observed on a transmission electron microscope (Hitachi H-7500). The size of exosomes was detected by Nanosight ns300 (Malvern Instruments Ltd., UK). For exosomes labeling, exosomes were labeled using PKH67 membrane dye (Sigma). Labeled exosomes were collected by ultracentrifugation after washing in 10 mL PBS, and resuspended in PBS. For cell treatment, exosomes were incubated with recipient cells for 48 h. An ExoQuick ULTRA EV Isolation Kit for Serum (System Biosciences) was used to isolate the exosomes from the serum of RCC patients. The RNAs in exosomes were extracted using an Exosome RNA Purification Kit (EZBioscience, USA).

For immunofluorescence analysis of EV uptake, the membrane of EV-donor A549 cells was stained with the PKH67 membrane dye (Sigma-Aldrich Chemie GmbH) according to the manufacturer’s recommendations. The resulting dyed EV pellet was prepared as described above and incubated with recipient THP-1 cells for 1 or 3 h, respectively. Cells were fixed for 15 min with 4% paraformaldehyde and nuclei were stained by addition of DAPI. Pictures were taken on an Axio Vert.A1 Fluorescence Microscope with an AxioCam MRm (Zeiss, Oberkochen, Germany).

FBS was ultracentrifuged at 100,000 g for 70 min to remove bovine EVs. Cells were cultured for 3 days in media supplemented with 0.5% EV-depleted FBS for EV isolation. Then the cell culture medium was sequentially centrifuged at 500 g, 2500 g and 12,000 g and the supernatant was collected. After ultracentrifugation at 100,000 g for 70 min, pellet was isolated and resuspended in 20 mL PBS. Then EVs were isolated from PBS solution using Total Exosome Isolation Reagent (Invitrogen 4,478,359).
Plasma EVs of mice were isolated. Blood was collected into an EDTA-K2 anticoagulant tube and mixed immediately to avoid clotting. Blood was sequentially centrifuged at 1500 g and 2400 g for 10 min at 4 °C, the supernatant was collected and diluted at ratio 1:1 with PBS. Then EVs were isolated using Total Exosome Isolation Reagent according to the manufacturer’s instruction.
Purified EVs were labeled with PKH67 membrane dye (Sigma-Aldrich) according to the manufacturer’s protocol. Briefly, EVs (50 μg) were suspended in 1 mL PBS before 1 mL Diluent C was added. Meanwhile, 4 μL PKH67 was added to 1 mL Diluent C and mixed gently with the EV solution for 4 min. Then 2 mL of 1% BSA/PBS was added to bind excess dye. Fluorescently-labeled EVs were then washed with PBS and re-extracted with Total Exosome Isolation Reagent.

The bMSC culture supernatant was collected and sequential centrifuged at 500 × g (10 min), 3,000 × g (20 min), and 10,000 × g (60 min) to remove impurities and then ultracentrifuged at 120,000 × g for 70 min (XPN-100, Beckman, USA) to precipitate the EVs. The purified EVs were placed on a carbon-coated copper mesh (90 s) and were stained with uranyl acetate dye solution (30 s) and then observed under a transmission electron microscope (Hitachi H-7500). Nanosight NS5300 (Malvern Panalytica, UK) was used to measure the particle size distribution based on a nanoparticle tracking analysis (NTA). EV surface marker protein levels were further measured by WB. The purified EVs were labeled using PKH-67 membrane dye (Sigma–Aldrich, USA) under the manufacturer’s protocol.

For the exosome uptake assay, PKH67 membrane dye (Sigma, USA) was added to PBS at a 1 µM concentration and used to label exosomes for 20 min before washing. Excess dye was removed by an additional washing step, and labelled exosomes (10 µg) were resuspended and used to treat THP-1 cells. After a 48-h incubation, THP-1 cells were then labelled with phalloidin and imaged by confocal microscopy.

Exosomes from CM and plasma were obtained as previously described [30 ], fixed with 2% paraformaldehyde, and placed on a 200 mesh Formvar-coated grid. This grid was stained with 2% phosphotungstic acid for 2 min, after which the exosomes could be observed. For fluorescent staining, PKH67 membrane dye (Sigma) was used to label exosomal membranes. Labelled exosomes were cleared by supercentrifugation and incubated with HUVECs for 6 h before the cultures were observed, as previously described [31 (link)]. The size and number of exosomes were detected by nanoparticle tracking analysis (NTA) using a NanoSight NS500 instrument (Malvern Panalytica, UK). A 488 nm laser camera was equipped to track Brownian motion and particle size for 60 s by producing a video. The videos were analysed using the NTA2.3 software. The concentration of exosomes was tested by BCA protein Assay Kit (Termo Scientifc, Rockford, lot. 23,235, USA). The surface marker proteins of exosomes were detected using WB.