2.12. SARS-CoV-2 Viral Load Quantified via 50% Median Tissue Culture Infectious Dose (TCID50) Infectious Viral Load Assay (TCID50)

Vero TMPRSS2 cells were seeded overnight at 25,000 cells/well at 37 °C, 5.0% CO₂ to achieve 80–100% confluent. The medium was aspirated and replaced with 180 µL of 1X DMEM supplemented with 2% FBS and 10 ug/mL gentamicin. Twenty (20) µL of the sample was added to the top row in quadruplicate and mixed with a pipette. Using a pipette, 20 µL was transferred to the next row and repeated down the plate (columns A-H), representing 10-fold dilutions. The tips were disposed of for each row and repeated until the last row. Positive (virus stock) and negative (media) controls were included in each assay. The plates were incubated at 37 °C, 5.0% CO₂ for 4 days. The cell monolayers were visually inspected for cytopathic effects (CPE). Uninfected wells had a clear confluent cell monolayer while infected wells displayed significant virus-induced cytopathic effects (CPE). Uninfected wells had a clear confluent cell monolayer while infected wells had little cell presence. The presence of CPE was marked on the lab form as a “+” and the absence of CPE as “0”. The TCID₅₀ value was calculated using the Reed–Muench formula.