Exo fect exosome transfection reagent

To assess the uptake of SCLC-cell-secreted exosomal miRNA in human umbilical vein vascular endothelial cells (HUVECs), Cy3-labeled miR-375-3p mimics were transfected into H446 cells, and the old culture medium (CM) was refreshed with CM containing exosome-depleted FBS for cell culture after 8 hours of transfection. Forty-eight hours later, exosomes in CM were collected by ultracentrifugation, followed by processing for PKH67 (Sigma) labeling. After washing with PBS once to remove excess dye, PKH67-labeled exosomes were harvested by ultracentrifugation and added to the CM of HUVECs. After incubation with PKH67-labeled exosomes for 12 hours, HUVECs were washed with PBS twice to remove excess exosomes followed by cell fixation using 4% paraformaldehyde solution. Cell nucleus of HUVECs were stained by DAPI and the uptake of exosomes in HUVECs was observed under a fluorescence microscope. All steps were conducted in the dark to avoid fluorescence quenching.
To transfer the mimics or inhibitors of miR-375-3p directly into isolated exosomes, Exo-Fect Exosome Transfection Reagent (System Biosciences, cat. NO. EXFT20A-1) was applied following the instructions.

For the engineering of EVs, Klotho recombinant protein was loaded onto EVs using the Exo-Fect Exosome Transfection Reagent (System Biosciences), according to the manufacturer’s protocol. In brief, 5 μg of EVs (quantified by Bradford) was incubated (by mixing) with 2.5 μg of recombinant Klotho in the presence of Exo-Fect solution for 1 h at +4°C. For the blockage of loading reaction, 30 μL of ExoQuick-TC was added to the mixture and kept for 30 min on ice. A pellet of Klotho-loaded EVs was obtained by a further 10 min of centrifugation at 13,000 rpm.