Exoquick tc

EVs from P17 and P20 UFs were isolated with the addition of exosome precipitation solution (Exo-Quick-TC, System Biosciences, Mountain View, CA, USA), which were incubated overnight at 4 °C according to the manufacturer’s instructions. The UFs with Exo-Quick-TC were then centrifuged at 1500 x g for 30 min at 4 °C. EVs were suspended and their protein concentration adjusted either in PBS (1 µg/µl) for TEM analysis and in vitro culture experimentations, whereas EVs were suspended in mammalian protein extraction reagent (M-PER, Thermo Fisher Scientific, Waltham, MA, USA) for western blot analysis^{26 (link),27 (link)}.

EV isolation and characterization were previously described [14 (link),15 (link)]. Briefly, NK3.3 cells were cultured overnight in RPMI-1640 media with 3% EV-depleted FBS and 200 U IL-2/mL and then treated with phorbol myristate acetate and ionomycin for 5 h to augment EV release. HEK293 cells were grown to 90% confluency over 48 h in DMEM and 3% EV-depleted FBS. NK3.3 cell suspensions and HEK293 culture media were centrifuged at 300× g for 10 min to pellet cells and debris. Supernatants were removed, centrifuged again at 2000× g for 10 min to remove smaller debris, and passed through a 0.22 μm filter. A commercial polyethylene glycol polymer for EV precipitation was added to the supernatants for a minimum of 12 h at 4 °C (ExoQuick-TC, System Biosciences, Palo Alto, CA, USA). After incubation, supernatants were centrifuged at 3000× g at 4 °C for 10 min per a modified ExoQuick-TC protocol. EV pellets were resuspended in phosphate-buffered saline (PBS). Protein concentrations were assessed using the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA); particle numbers and sizes were determined by nanoparticle tracking analysis (NTA) (NanoSight NS3000, Malvern Panalytical, Malvern, UK). There was minimal batch-to-batch variation in EV preparations made by PEG precipitation. For both HEK293 and NK3.3 EVs, 1 μg of protein consisted of 1–2 × 10¹¹ EV particles.

The sEVs from the cultivation media were isolated using the Complete SeraMir Exosome RNA Amplification kit (System Biosciences, Mountain View, CA, USA) containing Exoquick-TC (System Biosciences, Mountain View, CA, USA), using the protocol recommended by the supplier. Briefly, CM was centrifuged at 3000× g for 15 min to remove cells and cell debris. Then, 5 mL of supernatant was mixed with 1 mL of Exoquick solution, mixed, and then incubated overnight at 4 °C. Subsequently, the tubes were centrifuged at 16,000× g for 2 min, and the sEVs pellet was reconstituted in 350 μL of lysis buffer. After vortexing, followed by incubation for 5 min at room temperature to allow for complete lysis, 200 μL of 100% ethanol was added, and 600 μL of the mixture was filtered through the spin column at 16,000× g for 1 min. Two wash steps were then performed with 400 μL of wash buffer, followed by centrifugation at 16,000× g for 1 min, which was then followed by the elution of total RNA into 30 μL of elution buffer. The quality of the eluted exoRNA was then evaluated using the Agilent RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA, USA).