300 mesh copper grid

The visualization of purified VLPs involved uranyl acetate negative staining. Initially, 5 µL of the sample at a concentration of 1 mg/mL was adsorbed onto carbon formvar-coated 300 Mesh Copper grids (Agar Scientific, Stansted, UK), with the preparation of 2 grids per sample. After a 3 min incubation, the grids underwent washing with 1 mM ethylenediaminetetraacetic acid (EDTA) and subsequent negative staining using a 0.5% uranyl acetate aqueous solution. Analysis was carried out using a JEM-1230 electron microscope (JEOL, Tokyo, Japan) operating at an accelerating voltage of 100 kV. A minimum of five micrograph pictures were captured for each sample.

HCT-116 and RKO (control and treatment) cell lines were centrifuged and the culture medium above the pellets was replaced with 2.7% glutaraldehyde (Electron Microscopy Sciences, Hatfield, USA) in 0.1 M phosphate buffer, pH 7.4. Prefixation was performed at 4 °C for 2 h. The pellets were washed four times with 0.1 M phosphate buffer, and then postfixed for 24 h at 4 °C with 1.5% OsO₄ (Sigma-Aldrich) in 0.15 M phosphate buffer, pH 7.4. The cells were embedded in EMBed-812 (Electron Microscopy Sciences, Hatfield, USA), after a previous dehydration in an acetone series. Polymerization of the resin was performed at 60 °C for 72 h. Ultrathin sections were cut with a DiATOME diamond knife (DiATOME, USA) on a Bromma 8800 ULTRATOME III ultramicrotome (LKB, Sweden). They were collected on 300 mesh copper grids (Agar Scientific Ltd., Stansted, UK) and double contrasted with saturated alcoholic uranyl acetate (Merck, Darmstadt, Germany) for 12 min, and 2.8% lead citrate (Fluka AG, Buchs, Switzerland). The sections were examined on a JEOL JEM 1010 transmission electron microscope (JEOL Ltd., Japan) at 80 kV, and images were captured using a Mega VIEW III camera (Olympus, Soft Imaging System, Germany).