Pkh67 kit

Labeling of EVs was carried out according to the PKH67 kit (Sigma-Aldrich, St. Louis, MO, USA) instructions. In brief, 100 μL EV resuspension or PBS were added to 1 mL of Dilution C, respectively, followed by 4 μL PKH67 incubation for 4 min. Subsequently, the sample was closed with 2 times the volume of 0.5% BSA and leveled with PBS, then centrifuged at 120,000× g for 2 h at 4 °C. The precipitate was PKH67-labeled EVs.
Cells were fixed with 4% paraformaldehyde (Shyuanye, Shanghai, China) after coincubation with PKH67-labeled EVs or PBS with PTr2 cells for 48 h; the PTr2 cells were stained with TRITC Phalloidin (YEASEN, Shanghai, China) followed by DAPI staining according to the instructions. Subsequently, photographs were taken after 48 h of co-incubation using confocal microscopy (LSM 800, Zeiss, Oberkochen, Germany).

PKH67 Kit (Sigma-Aldrich, cat. PKH67GL, St. Louis, MI, USA) was used for the labeling of EVs. 100uL EVs were mixed with 0.4uL PKH67 reagent at room temperature, 200uL EVs-free FBS was used to stop the staining, the labeled EVs were obtained by centrifugation (120 000xg, 70min, 4°C) and resuspended in 100uL PBS. The concentration and size distribution of EVs were measured by Nano-particle analysis (ZetaView, PMX 110, Germany), Western blot assay was used for characterization of EVs related protein biomarkers. The microstructure of EVs was observed by a transmission electron microscope (Hitachi, Tokyo, Japan) using 1% (v/v) urayl acetate staining.

1 × 10⁶ MC3T3-E1 cells were plated in a six-well plate for culturing and 100 μL of FBS EXO or FBS EXO-ICA were added. 100 μL of the A and B solutions in the PKH67 kit (Sigma, United States) were prepared at a ratio of 1:4000, mix 200 μL, and incubated for 15 min at room temperature in the dark. 200 μL of 1% BSA were added to the above solution, which was centrifuged at 100,000 × g for 2 h. The concentration was measured, and the solution was added to the cells at a concentration of 20 μg per well, and cultured for 24 h. The cells were fixed with 4% paraformaldehyde for 20 min, and blocking solution was added for 20 min. Phalloidin (1: 200) was incubated in a wet box at 4°C overnight. Then the cells were stained with DAPI for 8 min prior to observation with an inverted fluorescence microscope.

EVs were purified from the cell culture supernatant of SKP-SCs that were cultured in differentiation medium. After the cell density reached 80% confluence, medium was switched to serum-free medium for 48 h. The supernatant, namely conditioned medium, was collected and went through sequential ultracentrifugation at 500g for 10 min to remove the cell debris, followed by filtering through a 0.22-μm filter (Millipore). EVs were isolated using the exoEasy Kit (QIAGEN) from 15 ml cell culture supernatant; the detail experiment steps were following the manufacturer’s protocol.
The morphology of the EVs was observed under transmission electron microscope (TEM, Hitachi), and nanoparticle tracking analysis (NTA, German particle Metrix) was utilized to measure EV diameter and particle number. The protein concentration was measured using Bicinchoninic Acid (BCA) protein assay (Thermo Scientific), and exosomal markers CD9, CD63, and CD81 as well as tumor susceptibility gene 101 (TSG101) were detected by Western blot analysis.
Fluorescence labeling of EVs was performed according to the protocol previously described [24 (link)]. The PKH67 kit (Sigma) was used according to the instruction manual. The labeled EVs were concentrated by an Amicon Ultra 10 kDa tube (Millipore), and the EV uptake experiment was performed.

The exosomes secreted by macrophages were isolated using gradient centrifugation [29 (link)]. Briefly, macrophages were cultured for 48 hours in exosome-free medium. Then, the macrophage culture medium was collected and centrifuged at 1000 × g for 10 min, 3000 × g for 30 min, 6000 × g for 40 min, and 12,000 × g for 2 hours to remove the cell debris. The supernatant was collected and centrifuged at 100,000 × g for 4 h at 4°C to collect the exosomes in the pellet. The size and concentration of the exosomes were determined using transmission electron microscopy and a nanoparticle tracking analyzer. Evaluate the purity of exosomes by detecting the expression levels of exosome markers such as CD81, TSG101, CD9, and calnexin using western blotting. The exosomes were labeled with a PKH67 kit (Sigma, St. Louis, MO, USA) to detect the phagocytosis of exosomes by tumor cells.