High speed centrifuge

Sow body weight and backfat thickness was measured at d 85 and d 107 of gestation and again at weaning (d 28 of lactation). Backfat thickness was measured using ultrasound equipment (Renco Lean-Meatier; Renco Corporation, Manchester, MA, USA) at 65 mm right of the dorsal midline of the last rib. At farrowing, the total number of piglets born, the number of piglets born alive, and the number of piglets born dead were recorded for each sow. The piglets were weighed within 24 h after farrowing and again at weaning (d 28 of lactation).
A 10 mL sample of the first colostrum of each sow was gathered from all the active mammary glands on the right side of the sow within one hour during farrowing. On d 14 of lactation, 20 mL samples of milk from each sow were collected approximately 30 s after sows were injected with 2 mL (equal to 10 units) of oxytocin intramuscularly in the hip (Ningbo Sansheng Biotech, Ningbo, China). The milk samples were collected into sterile tubes and immediately stored at −20 °C until further analysis. On d 0 and 28 of lactation, 10 mL of blood was collected from the ear vein of each sow, placed in heparin tubes, and centrifuged at 3000 rpm for 20 min by high-speed centrifuge (Thermo Fisher, Karlsruhe, Germany). The plasma was obtained, and samples were stored at −20 °C for later analysis.

A high speed centrifuge (Thermo Fisher Scientific Inc. USA) was used to centrifuge the medium and cell samples. Analytical balance (Mettler-Toledo, Switzerland), the Agilent HPLC instrument (Agilent Technologies, USA) equipped with an on-line Degasser Agilent 1260 Infinity, Agilent 1260 Bin pump, 1260 ALS automatic sample introduction system and an Agilent 1290 Thermostat temperature controller was used for the HPLC experiment. For detection of 5-HT, the equipment was connected to an Agilent 1260 DAD VL UV diode array detector. We actualized the chromatographic separation of 5-HT with an Agilent Zorbax Extend C18 Column (4.6 × 250 mm, 5 μm) under isocratic elution. The mobile phase is 0.05 mol/L KH₂PO₄ (apparent pH = 5)/acetonitrile (90:10, V/V), and the wavelength of the UV detector was set at 280 nm.

MSCs culture-supernatant containing the EVs was collected every 72 h starting at passage 2 up until passage 8, at an optical cell confluency higher than 90 %. The EVs were isolated using differential (ultra)-centrifugation steps as previously described [29 (link)] (Supplementary Fig. 1). Briefly, cell culture supernatant was centrifuged at 500 x g for 10 min to remove cells and cell debris (Hettich centrifuge, MA). To deplete large vesicles, samples were centrifuged again at 10,000 x g during 30 min and the final concentration of EVs was achieved by centrifuging the supernatant at 70,000 x g during 90 min (Thermo Scientific High-Speed centrifuge, Germany). All centrifugation steps were performed at 4 °C. The supernatant was discarded via aspiration. EV pellet was resuspended in 0.9 % NaCl or BV-2 cell culture medium and stored at -80 °C, depending on the application. Quantification of EVs was performed by flow cytometry or NanoSight nanoparticle tracking analysis (NTA).