Pkh26gl 1kt

Exosome labeling was performed using PKH26 red fluorescent cell linker kit for general cell membrane labeling according to the manufacturer’s instruction (PKH26GL-1KT, Sigma, China). PKH26-labeled exosomes were incubated with recipient cells for 24 h at 37 °C. After treatment, cells were washed with PBS twice, trypsinized using 0.25% trypsin-EDTA, and analyzed using a BD Caliber flow cytometer. The mean fluorescence intensity (MFI) was calculated according to the flow cytometry data.
For fluorescence imaging, cells were washed with PBS, fixed with 4% paraformaldehyde, permeabilized using 0.25% Triton, and stained with Alexa Fluor 488-labeled phalloidin in dark for 1 h followed by DAPI staining. Images were captured using a confocal laser scanning microscope with a × 40 objective (Leica DM IRB; Leica, Wetzlar, Germany).

The extracted exosomes from the HT29 cells were treated according to the PKH-26 protocols (PKH26GL-1KT, Sigma-Aldrich, St. Louis, USA). The exosomes were incubated with PKH-26-Diluent C staining solution for 5 min. Next, 2 mL of 10% BSA supplemented with PBS (D8537) was then employed to terminate the staining process. Following the addition of sucrose solution (1.5 mL), exosomes were centrifuged at 190,000 g for 2 h under conditions of 2-8°C in order to collect the pellet, with the medium and interface layer aspirated off. The exosomal pellet was pipetted to obtain the suspension in PBS, which was then transferred to the Amicon filter column. Following the addition of 9 mL of PBS as well as 0.75 mL medium, the exosome suspension was centrifuged at 3000 g for 40 min to reduce the volume to 0.5-1 mL [37 (link)].
The HMEC-1 cells were then incubated with PKH-26 labeled exosomes for 24 h, fixed by 4% paraformaldehyde at room temperature for 30 min, and stained with DAPI (36308ES11, Yeasen Company, Shanghai, China) for 5 min. The cells were then analyzed and photographed under an inverted fluorescence microscope (DMi8, Leica, Wetzlar, Germany).

BEVs were labeled with the red fluorescent agent PKH26 (PKH26GL-1KT; Sigma-Aldrich, USA) and centrifuged at 100 000 × g for 70 min to remove unbound dye. Then, the BEVs were suspended in PBS. We cocultured PKH26-labeled BEVs (500 μg/dish) with L929 cells. At the indicated times, we stained the cytoskeleton with phalloidine (49409-10NMOL; Sigma-Aldrich, USA; 5 μg/mL) and observed the cells by confocal microscopy (FV1000; Olympus, Tokyo, Japan). After being cultured for 3 days, the cells were stained with N-GSDMD and Hoechst 33342/PI according to the instructions. To further assess the effect of BEVs on pyroptosis and calcification, we added EVs (500 μg per dish), BEVs-depleted EVs (500 μg per dish) and BEVs (500 μg per dish) combined with a Caspase-1 inhibitor (Ac-YVAD-cmk, HY-16990, MedChemExpress, USA; 1 mg/mL dilution) to the cell culture system for 3 or 7 days. We assessed pyroptosis in the coculture system by performing green immunofluorescence staining for N-GSDMD after 3 days. To assess the effect of the BEVs on calcification, the system was maintained for 7 days. After being fixed with 4% paraformaldehyde, the cells were stained with Alizarin Red S (50 mmol/L, pH = 7.2). We removed the excess dye by washing the samples with water for 30 min. A Zeiss microscope (Thorn-wood, NY, USA) was used to image the plates. The stained areas were measured with ImageJ software.