Sigmafast reagent

The infectivity of OC43 was determined by focus-forming assay in HCT-8 cells and expressed as log₁₀ focus-forming units (FFU) per milliliter (Brown et al, 2019 (link)). Briefly, confluent monolayers of HCT-8 cells were incubated at 33°C for 1 h with 10-fold serial dilutions of virus. The cells were then washed and overlaid with infection medium containing 1% carboxymethyl cellulose (CMC). After 5 days of incubation at 33°C, the cells were fixed with 10% formalin, permeabilized with 0.1% Triton X-100, and blocked with phosphate-buffered saline (PBS) containing 1% bovine serum albumin and 0.1% Tween-20. OC43 antigen was stained with antibodies [primary: mouse anti-OC43 nucleoprotein antibody (MAB9013; Millipore, Burlington, MA, USA), secondary: goat anti-mouse horseradish peroxidase-conjugated antibody (Sigma-Aldrich, St. Louis, MO, USA)], visualized with SIGMAFast reagent (Sigma-Aldrich), and FFU were visually quantified.

The infectivity of OC43 was determined by focus‐forming assay in HCT‐8 cells and expressed as focus‐forming units (FFU) per milliliter [15 (link)]. Briefly, confluent monolayers of HCT‐8 cells were incubated at 33°C for 2 h with 10‐fold serial dilutions of virus. The cells were then washed and overlaid with infection medium containing 1% carboxymethyl cellulose (CMC). After 5 days of incubation at 33°C, the cells were fixed with 10% formalin, permeabilized with 0.1% Triton X‐100, and blocked with PBS containing 1% BSA and 0.1% Tween‐20. OC43 antigen was stained with antibodies (primary: mouse anti‐OC43 nucleoprotein antibody [MAB9013; Millipore, Burlington, MA, USA], secondary: goat anti‐mouse horseradish peroxidase‐conjugated antibody [Sigma‐Aldrich, St. Louis, MO, USA]), visualized with SIGMAFast reagent (Sigma‐Aldrich), and FFU were visually quantified. Values are the means of at least three independent determinations (with three replicates within each experiment).