Sw 32.1 ti rotor

For neuraminidase activity inhibition assays (NI) and STD-NMR experiments, hPIV-3 prepared by propagation in LLC-MK2 cells at a MOI of 1 were precipitated by treating clarified virus-containing supernatant of infection with a 5X solution of sterile 40%PEG-6000. Tubes were kept at 4 °C for 2 h under gentle agitation, centrifuged for 15 min at 3000 × g at 4 °C and the supernatants were discarded. The pellets were soaked at 4 °C overnight with 1/100^th of the initial volume of GNTE buffer (200 mM glycine, 200 mM NaCl, 20 mM Tris-HCl, 2 mM EDTA, pH 7.5). They were resuspended on the next day by gentle up-and-down pipetting. The precipitated virus was pooled and kept at 4 °C for later use in neuraminidase assay.
For STD-NMR experiments, the hPIV-3 particles were purified on a 30–60% non-linear sucrose gradient in GNTE buffer44 (link), as previously described19 (link). The PEG-precipitated virus was firstly resuspended in GNTE buffer using a dounce homogeniser, and loaded onto the gradient. The particles were purified by centrifugation at 100,000 × g for 2 h 30 without break at 4 °C, using a SW 32.1 Ti Rotor (Beckman Coulter, Brea, CA).

OptiPrep (Axis‐Shield, Oslo, Norway) density gradients were prepared as previously described (Tulkens et al., 2020a (link); Van Deun et al., 2014 ). Briefly, a discontinuous iodixanol gradient was prepared by layering 4 ml of 40%, 4 ml of 20%, 4 ml of 10% and 3.5 ml of 5% iodixanol in a 16.8 ml open top polyallomer tube (Beckman Coulter, Fullerton, California, USA). One millilitre of crude extract was placed on top of the gradient, followed by 18 h ultracentrifugation at 100 000 g and 4°C using a SW 32.1 Ti rotor (Beckman Coulter, Fullerton, California, USA). Fractions of 1 ml were collected and ODG fractions corresponding to a density of 1.04‐1.07 g/ml were pooled, as well as ODG fractions corresponding to a density of 1.09‐1.10 g/ml. SEC was performed on these pooled ODG fractions (identical to the first SEC) to remove iodixanol (Tulkens et al., 2020a (link); Vergauwen et al., 2017 (link)). SEC fractions 4–7 were pooled, concentrated to 100 μl, stored at ‐80°C until further use and referred to as the LPP extract (1.04‐1.07 g/ml) and EV extract (1.09‐1.10 g/ml).

OptiPrep™ density gradient (ODG) ultracentrifugation was conducted, as previously reported by Van Deun et al. [8 (link)]. Briefly, appropriate amounts of a homogenization buffer (10 mM Tris-HCl (tromethamine-hydrochloric acid), 1 mM EDTA (Ethylenediaminetetraacetic acid) and 0.25 M sucrose (pH 7.4)) and an iodixanol working solution were mixed in order to prepare 5%, 10%, 20%, and 40% iodixanol solutions. The iodixanol working solution was made by adding a working solution buffer (60 mM Tris-HCl, 6 mM EDTA, 0.25 M sucrose (pH 7.4)) to a stock solution of OptiPrep™ (60% (w/v) aqueous iodixanol solution). Moreover, the gradient was prepared in a 16.8 mL open-top polyallomer tube (Beckman Coulter) by layering 4 mL of 40%, 4 mL of 20%, 4 mL of 10%, and 3.5 mL of 5% solutions on top of each other. One mL of embryo-conditioned medium was overlaid onto the top of the gradient. Subsequently, the gradient was centrifuged at 4 °C for 18 h at 100,000× g (SW 32.1 Ti rotor, Beckman Coulter). Then, all 16 gradient fractions were divided into six samples by pooling the fractions 1–4, 5–7, 8–9, 10–12, 13–16, respectively. The pooled fractions were added to 14 mL PBS. Subsequently, the separate suspensions were centrifuged at 4 °C for 3 h at 100,000× g. The resulting pellets were resuspended in 100 µL PBS and stored at −80 °C for further EV characterization.

Extracellular vesicles isolated from FF and AOF were obtained via ODG UC [25 (link)]. Briefly, appropriate amounts of homogenization buffer (10-mM Tris-HCl, 1-mM EDTA, and 0.25-M sucrose (pH 7.4)) and iodixanol working solution were mixed to prepare gradients of 5%, 10%, 20%, and 40% iodixanol solutions. The iodixanol working solution was made by adding a solution buffer (60-mM Tris-HCl, 6-mM EDTA, and 0.25-M sucrose (pH 7.4)) to a stock solution of OptiPrep™ (60% (w/v) aqueous iodixanol solution). The gradient was prepared in a 16.8-mL open-top polyallomer tube (Beckman Coulter, Brea, CA, USA) by layering 4 mL of 40%, 4 mL of 20%, 4 mL of 10%, and 3.5 mL of 5% solutions on top of each other. The FF or AOF were overlaid onto the top of the gradient. Subsequently, the gradient was centrifuged at 4 °C for 18 h at 100,000× g (SW 32.1 Ti rotor, Beckman Coulter, Brea, CA, USA). Out of 16 layers of gradient fractions, EVs were mostly found in fractions 8 and 9 [25 (link)]; both fractions were pooled and diluted in 14-mL PBS and, subsequently, centrifuged for 3 h at 100,000× g and 4 °C. The resulting pellet was resuspended in 100-µL PBS and stored at −80 °C for further quantification and identification.