Sw32 rotor

Replication deficient VSV-G pseudotyped HIV-1 virions were produced in HEK293T cells using pCRV1-GagPol, pCSGW and pMD2.G as described previously (Price et al., 2014 (link)). At 24–48 hr post transfection, the supernatants were harvested and passed through 0.22 μm nitrocellulose filter. The virions were concentrated by ultracentrifugation through a 20% (w/v) sucrose cushion (2 hr at 28,000 rpm in a SW32 rotor [Beckman Coulter Life Sciences]). The pellet was resuspended in PBS, snap-frozen and stored at –80 °C. LEN-treated virions were incubated in presence of 700 nM LEN for 1.5 hr at room temperature prior to plunge-freezing for cryo-ET.

Overnight cultures of E. coli strain MRE600 were diluted 1:100 into 2–3 l of LB broth and grown to an OD₆₀₀ = 0.6. Cells were then pelleted and lysed by sonication. Ribosomes were pelleted and resuspended as described above for crude ribosome preparations. After resuspension in dissociation buffer, the 30S and 50S subunits were separated on a 15–35% (w/v) sucrose gradient in dissociation buffer on a SW-32 rotor (Beckman-Coulter) spun at 28 000 rpm (97 000 × g) for 16 h. Fractions containing the 50S and 30S subunits were collected separately, washed with dissociation buffer, and concentrated in a 100 kDa cut off spin filter. To increase purity, concentrated subunits were separately run on second 15–35% sucrose gradients and appropriate fractions were washed with dissociation buffer and concentrated.

Human cervical carcinoma cells (HeLa R19) were a gift from Dr G. Belov (University of Maryland, USA) and baby hamster kidney cells (BHK21, ATTC CCL-10) were obtained from the American Type Culture Collection (Rockville, MD). Cell lines were maintained in Iscove’s modified Dulbecco’s medium (IMDM; Lonza, Basel, Switzerland), supplemented with 10% fetal calf serum (FCS; GE Healthcare Bio-Sciences, Chicago, IL), 2 mM Ultraglutamine (Lonza), 100 U mL^-1 penicillin and 100 μg mL^-1 streptomycin (Gibco, Paisley, United Kingdom), in a humidified incubator at 37°C in an atmosphere with 5% CO₂.
EMCV stocks were obtained by transfection of BHK21 cells with in vitro RNA transcripts of the previously described infectious cDNA clone pM16.1, which contains a copy of the EMCV genome with a shortened poly-C tract [48 (link)]. Virus was harvested after observing virus-induced CPE and thereafter concentrated from cell-free culture supernatants by high-speed ultracentrifugation through a 30% sucrose cushion at 80,000xg for 16 hrs in a SW32 rotor (k-factor 321) (Beckman Coulter, Brea, CA).

The cell lysate was layered on top of a 14.5% v/v iodixanol (Opti-prep, Sigma) gradient and centrifuged at 28 000 rpm (96 300 × g) at 4°C for 18 h in a SW32 rotor (Beckman Coulter). The gradient was collected to 0.5 ml fractions on ice, using an Auto Densi-flow device (Labconco).

Vesicle preparations were performed in a similar manner to Zeev-Ben-Mordehai et al., 2014a (link). In detail, HEK293T cells were grown to confluency in T175 flasks using supplemented DMEM (1% non-essential amino acids (NEAA) (Sigma), 3% GlutaMax (GIBCO)) and 10% FBS (Sigma). Transient transfection was performed using 30 μg DNA of respective gB constructs and 185 μL polyethylenimine (PEI), each diluted in 4.5 mL supplemented, serum free DMEM which were mixed and added to cells in addition to 9 mL supplemented DMEM with 4% FBS. After 24 h the medium was exchanged for 18 mL supplemented, serum-free DMEM and cells incubated for another 24 h. 48 h post transfection cell supernatant was collected and replaced with 18 mL supplemented, serum-free DMEM. Supernatants were harvested every 24 h up to 120 h post transfection.
Vesicles were purified from cell supernatant by centrifugation at 4,000 xg for 20 min at 4°C to remove cellular debris. The pellet was discarded and the supernatant loaded in SW32 ultracentrifugation tubes before being underlaid by 5 mL of 20% sucrose solution in HEPES buffer (130 mM NaCl, 20 mM HEPES pH 8). After a 2 h spin at 30 000 rpm (∼150 000 xg) in a SW32 rotor (Beckman Coulter) at 4°C the supernatant was completely removed and discarded. Pelleted vesicles were rehydrated overnight in a small volume of HEPES buffer (100 μL / T175 flask).

Thawed plasma samples (volume 1.5–3 mL) were diluted with 0.9% NaCl (pH 7.4) to a final volume of 15 mL and filtered through a 0.22 µm filter. Filtered supernatants were centrifuged in an SW32 rotor (Beckman Coulter, Fullerton, CA, USA) at 100,000× g, for 14 h at 4 °C, to pellet EVs. EV pellets were washed in 0.9% NaCl (pH 7.4), centrifuged at 100,000× g for 2 h at 4 °C and resuspended in a volume of 0.9% NaCl. EVs were stored at 4 °C. EV size and concentration were further determined by Nanoparticle Tracking Analysis (NTA) using the NanoSight NS300 instrument (Malvern, Worcestershire, UK) with the scientific CMOS sensor. Briefly, EVs were diluted (1:500) in 0.9% NaCl. Three technical measurements were recorded under a controlled fluid flow with a pump speed set to 40 and a camera focus level adjusted between 10 and 16. The three videos were further analyzed using the NTA 3.1 Build 3.1.54 software to calculate the concentration, mode and mean size of EVs.

GC-EVs were isolated from the conditioned media of GC cell cultures, growing in EV-depleted medium, by differential centrifugation, as previously described [43 (link)]. Briefly, the conditioned media of each GC cell line was submitted to two sequential centrifugation steps (at 300× g for 10 min and 2000× g for 20 min) followed by a filtration step. The filtered supernatant was centrifuged at 100,000× g, 4 °C, in a SW32 rotor (Beckman Coulter, Fullerton, CA, USA) for 4 h to pellet EVs, and was further washed in PBS without Ca²⁺ and Mg²⁺ (Thermo Fisher Scientific) and centrifuged at 100,000× g, 4 °C, for 2 h. Each pellet containing GC-EVs was resuspended in an appropriate volume of 0.9% NaCl, in accordance with downstream applications.