Type 70 ti fixed angle titanium rotor

Placental samples were weighted, manually shattered with a scalpel, and cultured in vitro for 24 h in RPMI 1640 medium (Invitrogen Life Technologies, Carlsbad, CA, USA) 10% heat-inactivated ultracentrifuged FBS (Invitrogen Life Technologies, Carlsbad, CA, USA) at 37 °C and 5% CO₂. The culture medium was collected and subjected to subsequent centrifugation (1000× g for 10 min 4 °C, 2000× g for 20 min 4 °C, and 10,000× g for 30 min 4 °C) to remove cellular debris. Supernatant was ultracentrifuged in a type 70 TI fixed-angle titanium rotor (Beckman Coulter, Brea, CA, USA) at 100,000× g at 4 °C for 2 h. The pellet was suspended in PBS 1% DMSO (Sigma-Aldrich, St Louis, MO, USA) and stored at −80 °C for later use.

Twenty millilitres of 48 h conditioned media, per T175 (roughly 3 × 10⁶ cells at harvest, 80–90% confluency, >95% viability), was pre-prepared by a 300× g spin for 10 min at 4 °C to remove cells, a 2000× g spin for 20 min at 4 °C to remove cell debris and was filtered using a 0.2 µm filter to bias small EVs, <200 nm. This was placed into 25 mL polycarbonate tubes (Beckman Coulter, High Wycombe, UK) for ultracentrifugation using a Type 70 Ti Fixed-Angle Titanium Rotor (k-factor = 44, Beckman Coulter UK Ltd., High Wycombe, UK) at 100,000× g for 70 min at 4 °C [19 (link)]. Non-visible EV pellets were resuspended in 1 mL 0.2 µm filtered PBS before topping up the tubes for a second ultracentrifugation. After, the resulting pellet was resuspended in 60 µL of filtered PBS, snap frozen in liquid N₂ and stored at −80 °C for analysis to be completed within 1 month. We submitted all relevant data of our experiment to the EV-TRACK knowledgebase (EV-TRACK ID: EV230009) [47 (link)].

EVs were isolated from cell culture medium by ultracentrifugation as previously described (20 (link)). Briefly, when 70%–80% confluency was reached, cells were washed twice with phosphate-buffered saline (PBS; pH7.0) and then incubated for 48 h in FBS-free medium. Cell culture medium was collected and subjected to consecutive centrifugation steps (300 × g for 10 min and 2,000 × g for 20 min) to remove dead cells and cellular debris. The supernatant was vacuum filtered using a 10-kDa centrifugal filter (Merck Millipore, Darmstadt, Germany), and EV concentrates were ultracentrifuged at 100,000 × g for 70 min at 4°C (Type 70 Ti Fixed-angle Titanium Rotor, k factor = 157.4) (Optima™ XP ultracentrifuge; Beckman Coulter, Indianapolis, IN, USA). Pellets were washed with PBS followed by ultracentrifugation at the same speed and time. The supernatant was discarded, and the resulting EV pellets were suspended in PBS and stored at -80°C.
In order to obtain uEVs, urine samples from patients with PCa, BPH and HDs were centrifuged at 3,000 × g for 20 min at 4°C to remove debris and then ultracentrifuged at 200,000 × g for 2 h at 4°C. The supernatant was removed, and the uEV pellets were resuspended in PBS and stored at -80°C.

Wild-type ISKNV strains that had been repeatedly freeze-thawed^{53 ,55 (link)}, centrifuged, and filtered were centrifuged at 28,000 rpm at 4 °C for 1 h using Optima™ XE-100 with a Type 70 Ti Fixed-Angle Titanium Rotor (Beckman Coulter, USA). The precipitates, which were collected and resuspended in phosphate-buffered saline, were centrifuged at 150,000 g at 4 °C for 1 h using an SW 41 Ti Swinging-Bucket Rotor (SW 41 Ti) after being added to the upper layer of 35% sucrose (Sigma, USA) solution. Then, the supernatant was collected and centrifuged at 200,000 g at 4 °C for 1 h using SW 41 Ti after being added to the upper layer of a discontinuous sucrose solution density gradient (30%, 40%, 50%, and 60%, from the top to the bottom). The white suspensions located between 50% and 60% sucrose solutions were collected, resuspended in PBS, and centrifuged at 150,000 g at 4 °C for 1 h using SW 41 Ti. The final white precipitates are the purified virions.

Briefly, 3 × 10⁶ HEK293T cells were cultured on 10 cm plates with DMEM supplemented with 10% FBS, penicillin/streptomycin, and L-glutamine. In the next day, cells were transfected according to the manufacturer’s instructions using Lipofectamine 3000 transfection reagent (Invitrogen, USA) with shMMP14, psPAX2 and pMD2.G or psPAX2, pLenti-Luc and SARS-CoV-2 S plasmids for cotransfection. Supernatants were collected at 48 and 72 h post-transfection, passed through a 0.45 μm filter and subsequently centrifuged for 2 h at 70,000 x g using a Type 70 Ti Fixed-Angle Titanium Rotor (BECKMAN, USA) at 4 °C. Virus particles were resuspended and then aliquoted and stored at −80 °C. To infect target cells with SARS-CoV-2 virus pseudotypes, cells were seeded into 96-well plates, transfected with the indicated plasmids or siRNAs overnight, and then infected with SARS-CoV-2 virus pseudotypes for 72 h. Cells were washed three times, followed by lysis and detection of luciferase intensity using the Bright-Lumi™ Firefly Fluoresceinase Reporter Gene Assay Kit (Beyotime, CHINA).