Exoquick tc reagent

Exosomes were isolated utilizing a combination of centrifugation, ultracentrifugation, and filtration as we have previously described [16 (link)], or with the Exoquick-TC reagent (System Biosciences, Mountain View, CA, USA) following the manufacturer’s protocol. For ultracentrifugation isolation, conditioned cell culture medium was collected and centrifuged at 10,000 × g for 30 minutes at 4 °C, to remove cells and large debris. The supernatant was filtered using a 0.22-μm pore filter and the exosomes were pelleted at 100,000 × g for 1 h at 4 °C. The exosome pellet was washed with 10 ml of 1 × PBS and pelleted again by centrifugation at 100,000 × g for 1 h at 4 °C. The resulting pellet was either suspended in 1 × PBS for whole exosome applications or further processed for RNA or protein extraction. Plasma exosomes were isolated using the Exoquick reagent (System Biosciences, Mountain View, CA, USA) following the manufacturer’s protocol. The resulting exosome pellet was suspended in PBS and exosome concentration was estimated by Bradford assay.

To isolate exosomes, a T-MSC-conditioned medium (T-CM) was generated. Established T-MSCs [14 (link)] were grown to 80–90% confluence in 100 mm tissue culture plates, cells were washed four times with phosphate-buffered saline (PBS), and the medium was replaced with serum-free DMEM. The medium was collected after 48 h of culture, centrifuged at 1300 rpm for 5 min, and passed through a 0.2-μm filter. The CM was concentrated to 20-fold of the original concentration by centrifugal filtration (cut-off of 3K, Amicon Ultra-15, Millipore, Bedford, MA, USA). Thereafter, 1/5 volume of ExoQuick-TC reagent (System Biosciences, Palo Alto, CA, USA) was added to T-CM and mixed by vigorous inverting. After incubation at 4 °C overnight, the mixture was centrifuged at 1500× g for 30 min at 4 °C. The supernatant was removed, and final centrifugation was performed for 5 min at room temperature. The visible exosome pellet was resuspended in PBS, quantified using the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA), and stored at −80 °C.

When cells seeded in a 60 mm dish reached 75% confluency (~2.5 × 10⁶ cells), the supernatant media were collected and pre-cleared of cell debris by centrifugation at 2500 rpm for 10 min at 4 °C. Exosomes were isolated from the pre-cleared supernatant culture media of cells using ExoQuick TC reagent (System Biosciences, EXOTC50A-1, Palo Alto, CA, USA), according to manufacturer’s instructions. Briefly, 1 mL of reagent was added per 5 mL of culture media, and incubated at 4 °C for at least 12 h. Exosomes present in the incubated media were then pelleted down by centrifugation at 1500× g for 30 min and resuspended in either 50 µL of PBS or TRIzol reagent (Sigma, St. Louis, MO, USA) for RNA isolation or in RIPA protein lysis buffer for Western blot, and stored in −80 °C until further use. Serum exosomes were isolated from 250 µL serum using ExoCap composite kit (MBL International, Woburn, MA, USA) per instruction manual, which is based on an antibody coupled magnetic capture bead-based procedure. The kit contains a mixture of CD9, CD63, CD81 and EpCAM capture beads. This step was followed by the purification of exosomal RNA using ExoCap Nucleic acid elution buffer (MBL International, MEX-E kit, Woburn, MA, USA), according to the kit protocol.

BMSCs were cultured in DMEM (Hyclone, Logan, UT, USA), supplemented with 10% exosome-free fetal bovine serum (Exo-FBS; System Biosciences, Palo Alto, CA, USA), and 1% penicillin-streptomycin (P/S). Then, the cells were incubated at 3% O₂ in a three-gas incubator for 48 h. The medium was collected after incubation, centrifuged at 3000× g for 15 min, and passed through a 0.22 μm filter to remove dead cells and debris. The clarified conditioned medium was then size fractionated and concentrated through an Amicon^® Ultra-15 100 kDa centrifugal filter (UFC9100, Sigma-Aldrich, St. Louise, MO, USA) and centrifugation at 4000× g for 20 min. Next, the Exo Quick-TC reagent (EXOTC10A-1, System Biosciences, Palo Alto, CA, USA) was used to isolate exosomes, following the manufacturer’s instructions. The exosome-containing pellets were resuspended in phosphate-buffered saline (PBS) and stored at −80 °C until further use. A bicinchoninic acid assay (BCA) kit (Beyotime, Suzhou, China) was used to assess the total protein concentration of exosomes. Transmission electron microscopy and NanoSight analysis were used to observe the morphology and measure the size of the exosomes. Exosome markers, including Alix, CD9, and CD63, were analyzed via Western blotting.

Cells were cultured under serum-free medium (Invitrogen, Carlsbad, CA). The culture medium was harvested after 48 h of incubation, and the extracellular vesicles fraction was purified using Exoquick-TC reagent (System Biosciences, Mountain View, CA) according to the manufacturer's instructions. Extracellular vesicles were observed under a Tecnai T10 transmission electron microscope (FEI, USA).

Exosomes from the 24 h stimulated monocytes were extracted using ExoQuick-TC reagent (System Biosciences, Palo Alto, CA, USA). The exosomes were quantified on a Nanosight LM10 instrument (Malvern Instruments, Malvern, UK) as described in our previous report^{2 (link)}. The isolated exosome suspension in sterile PBS was added to HBMECs (1 × 10⁴ exosomes per 1 × 10⁵ cells) and cells incubated for 48 h before western blot analysis.