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Plaque assay protocol for viral quantification

ZM Zachary P. Morehouse
CP Caleb M. Proctor
GR Gabriella L. Ryan
RN Rodney J. Nash
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HcoV-229E was quantified with standard plaque assay protocols [19]. HEK-293 cells (ATCC, Cat. No. CRL-1573) were seeded at 2.0 × 105 cells per well in 6 well tissue culture treated plates (Fisher Science, Cat. No. 07–200-601) with 2 mL of DMEM (Fisher Scientific, Cat. No. 11–965-118) infused with 5% heat inactivated fetal bovine serum (Gemini Bioproducts, Cat. No. 100–500). Once the cells achieved 85% confluence, 200 μL of HcoV-229E stock was added to the media of the first well. The media was gently mixed and 200 μL of the infected media was transferred into the adjacent well. This was repeated to create serial dilutions throughout the plate. After 24 h of incubation, the virally infected media was removed from each well and replaced with 2 mL of DMEM infused with 5% heat inactivated FBS and 2% agarose (Bio-Rad, Cat. No. 1613101). The plate was incubated for an additional 5 days at 35 °C with 5% CO2 and plaques were counted to determine viral concentration in plaque forming units/mL (PFU/mL).

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