Exo fect

Exosomes were isolated from cell culture media using ExoQuick-TC according to the manufacturer’s instructions (System Biosciences, Palo Alto, CA, USA) and stored at −80 °C until use.
To examine the uptake of exosomes by cells, exosomes were first labeled using the ExoGlow™-protein EV labeling kit (System Bioscience). The cells were treated with the labeled exosomes for 24 h, fixed using paraformaldehyde (Sigma, St. Louis, MO, USA), and imaged using a confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).
Transfection of the miR-NC or miR-205-5p mimics into exosomes was performed using Exo-Fect™ according to the manufacturer’s protocol (System Biosciences).

CDC‐EVs were transfected with EV‐YF1 or EV‐YF1‐fluo (5′‐linked Rhodamine Red™‐X [NHS Ester], IDT) using Exo‐Fect (System Biosciences).

As mentioned above [21 (link)], we used Exo-Fect™ (System Biosciences, Palo Alto, USA) to load miR-494. First, miR-494 (100-300 pmol), exosomes (200 μg in 50 μL), and Exo-Fect were mixed and incubated at 37°C for 20 min (for the Control group, only miR-494 and exosomes were mixed without adding Exo-Fect), then ExoQuick-TC reagent (System Biosciences, Palo Alto, USA) was used to culture the mixture on ice for 20 min. Finally, exosomes/Exo^miR-494 were centrifuged at 13,000 × g for 5 min to collect and resuspend in phosphate buffer saline (PBS, Gibco™, USA) for reverse transcription (qPCR).

s.END1 EC-EVs were labeled with C. elegans cel-miR-39-3p transcript (Exiqon) using an exosomal transfection kit (Exo-Fect, System Bioscience) according to the manufacture’s instructions. EC-EVs were then washed by ultracentrifuging at 120,000 g for 120 minutes in 13 ml PBS. Labeled EC-EVs were injected into mice by tail vein injection at a concentration of 1 × 10⁹ EVs, and tissues of interest harvested. Tissues were perfused with heparinized PBS (10 ml) through insertion of a 25-gauge needle into the left ventricle. Tissues and plasma were harvested and snap frozen for qPCR analysis; harvested tissues were mechanically homogenized. To determine the uptake of labeled EVs by resident splenic cell populations (splenic monocytes vs. splenic ECs) isolated spleens were dissociated and splenocytes were incubated with anti-CD31 –conjugated (clone MEC 13.3, BD Pharmingen) magnetic beads (sheep anti-rat IgG Dynabeads, Invitrogen) to isolate splenic ECs as previously described (70 (link)). The resultant supernatant containing unbound cells was enriched for monocyte populations using EasySep (Stemcell Technologies) as previously described (1 (link)). Isolated cells were lysed and prepared for miRNA RT-qPCR.

Exosomes isolated from HOK were transfected using Exo-fect (System Biosciences, Mountain View, CA, USA) as per manufacturer’s instructions. Briefly, 100 µg exosomes were transfected with 20 nM of v-miR or control mimics using 10 µL Exo-fect and incubated for 37°C for 10 min. Reaction was stopped by adding ExoQuick and exosomes were pelleted after 30 min by centrifuging at 17,000 × g/3 min. Exosomes were resuspended in 500 µL serum-free media and incubated with the recipient cells at two different concentrations (100 and 250 µL) overnight before performing functional assays.