2.6. Viral Plaque Assay for Statistical Analysis

Day 1: Seed 6 × 10⁵ of each cell line (wild-type CV-1, CV-1-pTet-TurboFP635-EF-1a-Egfp, and CV-1-EF-1a-TurboFP635) with 2 mL 10% FBS DMEM per well in a 6-well plate to reach 100% confluence the next day. Day 2: Thaw an aliquot of vaccinia virus Lister 1.1.1 and sonicate at full power for 1 min in ice water (repeated three times), and dilute the virus to 10–6. Then, remove the medium from the cell culture wells, infect the cells with 250 µL viral solution per well, incubate the plate at 37 °C, 5% CO₂ for 2 h, and gently shake the plate every 20 min. Aspirate the infection media, add 2 mL of 5% FBS CMC culture media and incubate at 37 °C, 5% CO₂ for another two days. Day 4: Remove the CMC medium, stain the plates with 1 mL per well crystal violet for 3–5 h/overnight at room temperature, remove the stained medium into an extra glass bottle (for later sterilization) and wash the cells with tap water, then count the number of viral plaques.