Sw 41 ti swing bucket rotor

Fetal bovine serum (FBS) was centrifuged at 35,000 rpm for overnight (>16 h) (Beckman SW 41 Ti swing-bucket rotor) to remove bovine-derived exosomes. The supernatant was filtered using a 0.22 μm filter. Extracellular vesicle-free FBS (EV-free FBS) was added to DMEM to make a final concentration of either 2.5% or 5% EV-free FBS DMEM without penicillin-streptomycin.

100 mg of liver tissue were lysed in 2.5 ml of lysis buffer A (10 mM KCl, 10 mM Tris–HCl pH 8.0, 2 mM MgCl₂ and 0.05% NP-40) with a protease inhibitor cocktail (Roche #04693116001). Following incubation on ice for 20 min, tissues were lysed using a tight-fitting douncer with 40 strokes, followed by centrifugation at 13 000 rpm spin at 4°C for 10 min to remove cell debris. A sucrose gradient was created by adding 2 ml of 36, 29, 22 and 15% sucrose to Beckman ultracentrifuge tubes and then 2 ml of the supernatant sample were added to the sucrose gradient to make up the final volume to 10 ml. A discontinuous gradient of 15–36% was formed and the tubes were centrifuged at 40 000 rpm for 5 h at 4°C (Beckman SW 41 Ti swing-bucket rotor). From the top of each gradient, 800 μl gradient fractions were collected to yield a total of 12 fractions. The Alexa fluorescence intensity of the fractions was measured using TECAN infinite M1000 Pro (TECAN).

To assay for the packaging efficiency of MCV fluorescent reporter viruses, cell lysate from 293 TRE-sTco cells that were transfected and harvested as described in virion production were overlayed on 9 mL discontinuous gradient (3 mL of 27%, 3 mL of 33%, 2 mL of 39% and 1 mL of 60% iodixanol) and subjected to ultracentrifuge at 41,000 rpm for 6 h at 16 °C in SW 41 Ti swing-bucket rotor (Beckman, Indianapolis, IN, USA). Twelve fractions (1 mL each) were collected from the top of the tube using a pipette. Fractions were used to perform immunoblotting, qPCR (in the presence or absence of Benzonase treatment) and an infection assay.
To assay for the packaging efficiency of the pseudovirus system, 293 TRE-sTco cells were co-transfected with 18 µg MCV genome DNA or reporter plasmid pEGFP-N1, 15 µg pWM plasmid (expressing VP1) and 5 µg ph2m plasmid (expressing VP2) in T75 flasks. Three days after transfection, cells were harvested and virions were purified as described above.