Edta coated tubes

Blood samples of overnight fasted sows were taken at the first day of lactation (D1), 2 weeks after the start of the feed restriction period (D17), at weaning (D24) and at slaughter (D26), between 06.00 and 07.00 h. The sows were restrained with a nose-sling, and blood was collected from the jugular vein in 9 mL serum clot activator tubes and in 9 mL EDTA coated tubes (Greiner Bio-One, Monroe, NC) to obtain serum and plasma samples, respectively. The collection tubes were stored on ice. After blood sampling, the EDTA tubes were immediately centrifuged, while the serum tubes were first incubated for 1 h at 4 °C. Centrifugation took place at 3000×g for 10 min at 4 °C. Both plasma and serum samples were stored at −20 °C until further analysis.

Blood was collected by retro-orbital bleeding into EDTA-coated tubes (450532; Greiner Bio-One, Kremsmünster, Austria) and kept on ice. Plasma was separated by centrifugation and 5 mg reduced glutathione (G4251; Sigma–Aldrich, St. Louis, MO, USA) was added per mL of plasma after centrifugation to prevent degradation of epinephrine and norepinephrine. Plasma was snap-frozen and stored at −80 °C until further use. Corticosterone, epinephrine, and norepinephrine were measured by online solid phase extraction (SPE) in combination with isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS). Corticosterone measurement was performed essentially as described by Hawley et al. [28 (link)], using corticosterone-D4 as a stable isotope-labeled internal standard on an online SPE manager in combination with a XEVO TQ mass spectrometer (Waters, Milford, MA, USA). Epinephrine and norepinephrine were analyzed as described by Van Faassen et al. [29 (link)], using epinephrine-D3 and norepinephrine-D6 as stable isotope-labeled internal standard on a Symbiosis Pharma online SPE system (Spark Holland, Emmen, The Netherlands) in combination with a XEVO TQ mass spectrometer (Waters, Milford, MA, USA).

Male C57BL/6J mice (Jackson Labs, Bar Harbor, ME, USA), 8 to 12 weeks of age, were used in this study. The animals were kept at a constant temperature (25 °C) under a 12 h light/dark cycle with free access to food and water. On the first and second day, animals received a 200 μL intraperitoneal injection of PBS or sonicated EVs at a concentration of 2.1 × 10¹⁰ particles/mL (by NTA). On the third day, animals received an intraperitoneal injection of 5 mg/kg LPS. Three hours later, animals were anesthetized and sacrificed via cardiac blood collection. Blood was collected into EDTA-coated tubes (Greiner Bio-One; Monroe, NC, USA) and spun at 1000× g for 15 min to produce plasma. All animal work was carried out in accordance with the NIH guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Maryland College Park.