10 cm petri dishes

When cells cultured in 10 cm petri dishes (Corning; NY, USA) reached 80–90% confluence, cell were collected, centrifuged, washed with PBS, and immediately fixed in 2.5% glutaraldehyde fixative solution for 2 h at room temperature. The cells were then further fixed with a 1% citric acid solution for 1.5 h in the dark followed by dehydration with series concentrations (50%, 70%, 80%, and 90%) of ethanol and embed in the resin overnight. Ultrathin sections of 70 nm thickness were cut with an ultramicrotome (UC7, Leica, Germany) and collected on 200-mesh copper grids. The sections were stained with 2% aqueous solution of uranyl acetate for 20 min in the dark and incubated in lead citrate solution for 7 min. Autophagosomes were examined with an electron microscope (Hitachi HT-7800) operating at 80 kV.

293T Lenti-X cells (3.5x10⁶ cells) were transfected on 10 cm Petri dishes (Corning) to produce each IDLV-CoV2 as described above. At 48 hrs post transfection, cells were stained with anti-Spike COVA2-15 mAb (47 (link)), with the exception of IDLV producing beta Spike, which was stained with COVA1-16 mAb (47 (link)), followed by Goat Anti-Human IgG H and L (10 nm Gold) used as a secondary Ab (Abcam, Cambridge, UK). After staining, cells were fixed with 2.5% glutaraldehyde in cacodylate buffer 0.1 M, pH 7.2. Fixed cells were washed and post-fixed in 1% OsO4 using the same glutaraldehyde/cacodylate buffer. Fixed specimens were dehydrated by using a graded series of ethanol solutions and then embedded using an Agar 100 resin (Agar Scientific, Essex, UK). Ultrathin sections were placed on 200-mesh copper grids and then stained with lead citrate and uranyl acetate. Sections were analysed by using a Philips 208S transmission electron microscopy (TEM) at 100 kV.

Before plasmid transfection, HEK293T cells were divided into five 10-cm Petri dishes (Corning, USA) and cultured for 1 day. Thereafter, the cells were co-transfected with the myc-tagged protein expression plasmids and flag-tagged plasmids, with pCMS-flag used as a control. After 24 h, the cells were harvested using PBS, the mixture was centrifuged at low speed, the supernatant was discarded, and the pellet was lysed lysed with cell lysis buffer (Beyotime, China). Next, 20 μL of the cell lysate was put aside as an input sample, and the remaining lysate sample was added to anti-flag M2 magnetic beads (Sigma). The mixture was shaken gently at 4°C for more than 2 h, following which the magnetic beads were rinsed three times using cell lysis buffer. The 20 μL input sample and co-immunoprecipitation (co-IP) sample (the rinsed magnetic beads) were then incubated with 2× protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer (Takara) at 100°C for 5 min. The target proteins were examined with the western blot assay using the anti-myc antibody (Transgen, China) and anti-flag antibody (Sigma).

Spheroids (3D cultures) were formed from monolayer eMSCs (2D cultures) using the hanging drop technique. Seven thousand cells per 35 μL of DMEM/F-12 medium containing 10% of FBS were placed in drops on the cover of 10-cm Petri dishes (Corning) and inverted. During the next 48 h, cells spontaneously aggregated in hanging drops and formed spheres, which were transferred then to dishes coated with 2-hydroxyethyl methacrylate (HEMA, Sigma) and cultured in 2 ml of full growth medium for 24 h.

VSV-based pseudotyped particles were produced as described previously35 36 (link). 9 × 10⁵ cells BHK-21 were seeded in 10 cm petri dishes (Corning), and incubated for 24 h. BHK-21 cells were transfected by a mixture containing 36 μL of lipofectamine 2000 transfection reagent (Thermo Fisher) and 12 μg of plasmid DNA (6 μg of HA- and NA-encoding plasmids DNA) for each plate, and incubated for 24 h. Next, transfected BHK-21 cells were inoculated with VSV *G-VSVΔG in RPMI medium (10.4 g RPMI powder, 26.7 mL BSA 7.5% liquid, 25 mL 1 M HEPES, 1 L H₂O), and incubated for 2 h at 37 °C with rocking. Unbound viruses were washed out with DPBS (Dulbecco’s phosphate-buffered saline), and incubated for 24 h at 37 °C with DMEM growth medium. For HA only case, 0.25 units of exogenous neuraminidase from C. welchii (Sigma-aldrich) was added to the plate to facilitate viral particle release. The supernatants, which contain pseudotyped particles, were collected after several gentle taps on the walls of petri dishes to help the release of particles. The supernatants were ultracentrifuged in a Ti45 rotor at 35,000 rpm for 120 min. The supernatants were discarded, and pellets were resuspended in 3 μg/ml trypsin and incubated for 30 min at 37 °C to activate HA. The trypsin-treated viral solutions were then aliquoted for storage at −80 °C.