Falcon 96 well black clear flat bottom tc treated imaging microplate

A total of 0.5–2.0 × 10³ cells/well were cultured for imaging on a Falcon® 96-well Black/Clear Flat Bottom TC-treated Imaging Microplate (Corning, New York, NY, USA) for 72 h. When cells reached the appropriate confluence, their surface was washed with a PBS solution, fixed in freshly prepared 4% (v/v) paraformaldehyde (Avantor Performance Materials Poland, Gliwice, Poland) for 10–15 min, washed with PBS, and permeabilized in 0.25% (v/v) Triton X-100 (Sigma‒Aldrich, Saint-Louis, MO, USA) for 15 min at RT. Then, the cells were washed and blocked for 30 min in 1% (w/v) bovine serum albumin (Sigma‒Aldrich, Saint-Louis, MO, USA) solution in 0.1% (v/v) PBS/Tween 20 (Sigma‒Aldrich, Saint-Louis, MO, USA) at RT. Next, the fixed cells were incubated with primary antibodies against vimentin (NBP1-31327, dilution 1:500; Novus Biologicals, Centennial, CO, USA) in a blocking solution at 4 °C overnight. Next, after washing with PBS, a secondary antibody (anti-rabbit antibody Alexa Fluor 488, ab150077; Abcam, Cambridge, UK) in the blocking solution was used for 1 h at RT. Finally, the samples were rinsed with PBS and photographed using an Olympus IX81 fluorescence microscope (Olympus, Warsaw, Poland) with CellSense software (Olympus, Warsaw, Poland).

For imaging, 10⁴ cells/well were cultured on a Falcon^® 96-well Black/Clear Flat Bottom TC-treated Imaging Microplate (Corning, New York, NY, USA), following the procedure described above. On the day of staining, cells were washed twice with PBS solution, fixed for 10–15 min in freshly prepared 4% paraformaldehyde (Avantor Performance Materials Poland, Gliwice, Poland), and permeabilized in 0.25% Triton X-100 (Sigma-Aldrich, Saint-Louis, MO, USA) for 15 min at room temperature. After two washes in PBS solution, cells were blocked for 30 min in 1% bovine serum albumin (Sigma-Aldrich, Saint-Louis, MO, USA) solution in 0.1% PBS/Tween 20 at room temperature. Then, the cells were rinsed with PBS three times (5 min each) and counterstained with DAPI (1:1500; Cell-Signaling, Danvers, TX, USA) and DyLight™ 554 Phalloidin (1:100; Cell-Signaling, Danvers, MA, USA) in PBS solution for 15 min at room temperature. Cells were reviewed and photographed under an Olympus IX81 fluorescent microscope (Olympus, Warsaw, Poland) with CellSense software (Olympus, Warsaw, Poland).

In order to quantify viral progeny in supernatants, 5 × 10³ Vero cells were seeded in 96-well plates (Falcon^® 96-well Black/Clear Flat Bottom TC-treated Imaging Microplate, Corning, Glendale, AZ, USA). The following day, virus-containing supernatant samples were serially diluted on cells by 10-fold and then incubated for five days. Cytopathic effects were visually inspected.