Pkh26 red fluorescent cell linker kit

Evs derived from RCC cells were labeled using the PKH26 Red Fluorescent Cell Linker Kit (Umibio, Shanghai, China). Following this, PMA-stimulated THP-1 cells [THP-1 (Mφ)] were co-incubated with these PKH26-tagged Evs in a dark setting overnight. After staining the cells with 4′,6-diamidino-2-phenylindole (DAPI), the EV uptake process was visualized using a confocal fluorescence microscope (Carl Zeiss AG, Jenna, Germany).

The EVs derived from RCC cells were labeled by using the PKH26 Red Fluorescent Cell Linker Kit (Umibio, Shanghai, China). Then MRC5 cells were incubated with the PKH26-labeled EVs in the dark overnight. After the cells were stained by 4′,6-diamidino-2-phenylindole (DAPI), confocal fluorescence microscopy (Carl Zeiss AG, Jenna, Germany) was used to observe the internalization of EVs.
For the internalization of circEHD2-ASO, Cy3-labeled circEHD2-ASO was incubated with human RCC cells (OSRC-2 and 786-O) in the dark for 24 h. Then, the PKH67 Green Fluorescent cytomembrane Linker Kit (Solarbio, Beijing, China) was used to label the cytomembrane of RCC cells at 4℃ for 20 min. Next, the cells were washed twice with phosphate buffered saline (PBS), before DAPI was used to stain the nuclei of the RCC cells. The internalization of circEHD2-ASO in RCC cells was photographed by confocal fluorescence microscopy (Carl Zeiss AG, Jenna, Germany). The sequence of circEHD2-ASO was described in the Table S2.