Bovine albumin

To measure the inhibitory activity of the extract and active compounds on the replication of HCoV-NL63, LLC-MK2 and Calu-3 cells were infected by HCoV-NL63, simultaneously treated with the extract and the active compounds, and then subjected to the assay according to the methods of the CPE reduction assay, as described above. Later, the cells were fixed with 4% paraformaldehyde in PBS for 30 min, quenched with 50 mM NH₄CI for 15 min, and permeabilized and blocked using 1% albumin bovine (Affymetrix) plus triton X-100 (Thermo Fisher, Waltham, MA, USA). After a 4 h incubation at 4 °C, the cells were reacted overnight with the 1/2000 dilution of HCoV-NL63-immunized sera in 1% BSA at 4 °C, and treated with 1/3000 dilution of Alexa Fluor anti-mouse IgG antibodies in 1% BSA for 1 h at 4 °C (Thermo Fisher). The cells were further stained with 4′,6-diamidino-2-phenylindole (DAPI) for 20 min at room temperature (Thermo Fisher). The image analysis of stained cells was recorded using fluorescent microscopy and the ImageJ2 software. The infectivity was represented as the ratio of HCoV-NL63-positive cells (red fluorescent signals) to total cells (blue fluorescent signals).