Ultra 15 centrifugal filter unit

The shRNAs for swine NLRP3 used in the study are listed in Table 1. The shRNA plasmids (pLenti-shNLRP3) were transfected into HEK293T cells together with pMDLg, pRSV-rev, and pCMV-VSV-G using Lipofectamine 3000 (Life Technologies). The ratio of pLenti-shNLRP3, pMDLg, pRSV-rev, and pCMV-VSV-G was 2:1:1:1, respectively. Culture supernatants were harvested at 24 h post-transfection (pt) and 48 h pt and were centrifuged at 4,500 × g for 20 min at 4°C to remove cellular debris. The recovered viruses were concentrated using an Amicon Ultra-15 centrifugal filter unit with an Ultracel-10 membrane (Millipore). The viruses were resuspended in RPMI-1640 medium, and the viral titers were determined using HEK293T cells. Monocytes were transduced with the same amounts of lentiviral particles encoding non-targeting control or gene-specific shRNA in the presence of 8 μg/mL polybrene. After 48 h, monocytes were infected with CSFV at 1 MOI. After another 12 h, the shRNA knockdown efficiency of the target protein in lentivirus-transduced cells was assessed by western blot analysis. The concentrations of IL-1β were measured by ELISA.

The CM was collected and centrifuged at 1000g for 5 minutes. The supernatant was centrifugated for 30 minutes using the Amicon^® Ultra-15 Centrifugal Filter Unit (3 KDa, Millipore, USA). Afterward, the concentrated liquor was mixed with an equal volume of 50% Trichloroacetic acid (TCA) solution and incubated on ice for 2 hours. Then, the protein precipitation was obtained after centrifuged at 2000g and 4°C for 5 minutes and washed twice with 1ml of pre-chilled (-20°C) acetone.

The tooth roots of porcine second incisors at 1 year of age were dissected to 6 mm in length and 2 mm in width, and were treated by the following process at 4 °C. The periodontal ligament was removed from the surface of the root by an excavator (nonextracted tooth). The teeth were demineralized with 0.6 M HCl for 7 days (HCl-extracted tooth), and further extracted with 4 M GdnHCl in 50 mM Tris–HCl, pH 7.4 for 7 days (GdnHCl-extracted tooth) [13 ]. They were then extracted with 0.5 M EDTA in 50 mM Tris–HCl, pH 7.4 for the next 7 days (EDTA-extracted tooth) [14 (link)]. The GdnHCl extracts and the EDTA extracts were concentrated respectively to 4 μg/ml by centrifugation with an Amicon Ultra-15 Centrifugal Filter Unit with an Ultracel-3 membrane (Millipore) and stored at –80 °C until use. After washing four times with phosphate-buffered saline (PBS, pH 7.4), the four distinct types of the tooth (eight teeth, respectively) were kept in PBS at 4 °C until use.

BCs were maintained in culture in DMEM/F12 medium supplemented with N2 and B27 (see above). After the specimens were delivered to the Esrange laboratory, the medium was collected for exosome analysis. Exosome isolation was performed using Amicon^® Ultra-15 Centrifugal Filter Unit with Ultracel-100 regenerated cellulose membrane (UFC910024, Millipore, Massachusetts, United States). The cellular medium was centrifuged at 2000 rcf for 30 min at 4°C and washed with PBS at 2000 rcf for 30 min at 4°C. Exosomes kept by the filter were then collected and stored at −20°C.

The sEV were isolated as described by Li et al. [26 (link)]. The sEV-free FBS was isolated as described by Théry et al. [27 ]. Before sEV isolation, FBS was centrifuged at 120,000g for 12 h, then the supernatant was obtained as sEV-free FBS. BMMSC and NRK-52E were cultured in DMEM/F12 containing 10% sEV-free FBS for 48 h. Then, the culture medium was centrifuged at 300g for 10 min to eliminate dead cells and was centrifuged at 3000g for 20 min to remove cell debris. The obtained supernatant was concentrated by an Ultra-15 centrifugal filter unit (Millipore, USA) and then centrifuged at 13,000g for 30 min to remove the microvesicles. Afterward, the supernatant was centrifuged at 120,000g for 70 min to concentrate the sEV. The pellet was washed with PBS by repeating the centrifugation conditions of the last step and was resuspended in a small volume of PBS. For the following experiments, the sEV were stored at − 80 °C. The morphology of sEV was identified by transmission electron microscopy (Hitachi H7500 TEM, Japan). The diameter was measured by NanoSight (Malvern Panalytical, UK). The surface markers of sEV, CD9 and CD81, were detected by a western blot. The protein content was quantified using a bicinchoninic acid protein assay kit (Beyotime, China).