Optima xl 100k ultracentrifuge

The exosome isolation was performed by modifying the protocol of Théry et al. [40 (link)] (protocol suggested by Miltenyi Biotec: DS MACSPlex Exosome Kit human 130-108-813). Briefly, a 1.3 mL plasma sample was diluted in an equivalent volume of phosphate buffered saline (PBS) and centrifuged at 2000× g for 30 min at room temperature. Subsequently, the supernatant was centrifuged at 10,000× g for 45 min at 4 °C (Sorvall RC-5B Superspeed Centrifuge, Du Pont Instruments, USA), and the new supernatant was ultracentrifuged at 100,000× g for 120 min at 4 °C (Optima XL-100K Ultracentrifuge, Beckman Coulter, Brea, CA, USA). Then, the precipitate was resuspended in a volume of PBS equivalent to the initial volume of plasma (1.3 mL) and filtered through 0.2 μm filters (Titan3™ PES (polyethersulfone) Syringe Filters, Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, the filtering was subjected to a new ultracentrifugation at 100,000 × g at 4 °C (Optima XL-100K Ultracentrifuge, Beckman Coulter, Brea, CA, USA). The exosome precipitate was resuspended in 50 μL of 5% sodium dodecyl sulfate (SDS)-PBS with antiproteases (cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail, Roche, Switzerland) and stored at −80 °C for further analysis.

MDCK cells were seeded into T75 flasks at 5 x 10⁶ cells per flask 24 h prior to infection. Each infection took place under synchronized, single-cycle conditions. Duplicate flasks were inoculated with GFHK99wt virus at an MOI of 1 GC/cell and either PBS, homologous GFHK99var virus, or heterologous MaMN99wt, NL09var, or Pan99wt viruses at an MOI of 8 GC/cell. At 24 h post infection medium (containing viral progeny) was collected separately from cells. Cells were washed 3x with PBS and harvested using the Qiagen RNeasy mini kit and protocol instructions. Collected medium was centrifuged (Thermofisher Sorvall ST 16R Centrifuge) at 3000 rpm for 10 min at 4°C to remove cell debris. The resultant supernatant was transferred into a Beckman unltracentrifuge tube and centrifuged (Beckman Coulter Optima XL-100K Ultracentrifuge) at 10,000 rpm for 30 min at 4°C to further clarify the sample of cell debris. The final supernatant was then transferred into a new unltracentrifuge tube. A cushion of 5 mL 30% sucrose in NTE buffer [1 M NaCl, 0.1 M Tris, 0.01 M EDTA, pH 7.4] as injected into the bottom of the tube and the sample was centrifuged (Beckman Coulter Optima XL-100K Ultracentrifuge) at 25,000 rpm for 2 h at 4°C. The resultant viral pellet was resuspended in PBS and RNA extracted using the Qiagen Viral RNA mini kit and protocol instructions.

Single gRNAs targeting Hdac5 gene were designed using tools available from the Genetic Perturbation Platform (Broad Institute) and cloned into lentiGuide-Puro plasmid. Lentiviral vectors (LV) were obtained transfecting 293T cells with a solution containing a mix of the selected LV genome transfer plasmid, the packaging plasmids pMDLg/pRRE and pCMV.REV, pMD2.G and pAdvantage, as previously described (Milani et al., 2019 (link)). Medium was changed 14 to 16 hours after transfection and supernatant collected 30 hours after medium change. Vector-containing supernatants were passed through a 0.22-μm filter, transferred into sterile polyallomer tubes and centrifuged at 20,000 x g for 120 min at 20°C (Beckman Optima XL-100 K Ultracentrifuge). LV pellet was dissolved in the appropriate volume of PBS to allow 500 × to 1000 × concentrations. 2.5 × 10⁵/well Cas9-expressing BMDMs were transduced twice at day 5 and 6 of differentiation with a multiplicity of infection (MOI) of 10 in L929-conditioned medium supplemented with polybrene (8 μg/ml). After the second hit, transduced cells were selected with puromycin (5 μg/ml) for 48 hours and then stimulated as indicated.