Evaluating Immune Response of Extracellular Vesicles

PMNs (120 μL of 2.5*10⁷/mL) were added to 480 µl of EV samples at 37°C in a linear shaker (80 rpm) for 3 hours. Cells were centrifuged (500 g, Hermle Z216MK 45° fixed angle rotor, 10 min, 4°C) and supernatants were analyzed for IL-8, TGF-β, IL-1RA, IL-1α, IL-6, TNF-α with a human IL-8, TGF-β, IL-1RA, IL-1α, IL-6, TNF-α DuoSet sandwich ELISA kit according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA) in a microplate reader (Labsystems iEMS Reader MF, Thermo Scientific). We also prepared a control sample for the oZ-EV samples that contained the same amount of zymosan as the oZ-EV isolates. To achieve this, in half of the oZ-EV batch EVs were lysed whereas zymosan particles were left intact (16 (link)). We refer to this sample as “lysed oZ-EV”.