2019 ncov spike s1 antibody

We performed a cell-based coronavirus immunostaining assay using a rabbit (2019-nCoV) spike S1 antibody (1:50; Sino Biological, Duesseldorfer, Germany) and goat anti-rabbit secondary antibody conjugated with Alexa Fluor 647 (A-21245; 1:1000; Invitrogen, Carlsbad, CA, USA), as previously described (Stefanik et al., 2021 (link)).

Transwell membranes were cut into small pieces and placed on positively charged microscope slides and blocked in blocking buffer (1% bovine serum albumin (BSA), 1% Tween20, and 22.52 mg/mL glycine) for 1 hour at room temperature. Following blocking, the membranes were incubated in SARS-CoV-2 (2019-nCoV) Spike S1 Antibody (Sino Biologicals, Cat# 40150-R007) and caspase-3 antibody (abcam, Cat# ab32351) at 1:250 dilution overnight at 4°C. The following day, glass slides were washed 3 times with sterile PBS and incubated in Alexa Fluor 488 goat anti-rabbit secondary antibody (abcam, Cat# ab150077) and Alexa Fluor 594 goat anti-rabbit secondary antibody (abcam, Cat# ab150080) at 1:500 dilution for 1 hour at room temperature in the dark. This was followed by PBS wash (X3) and staining for nuclei using 0.5 µg/mL 4’,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Cat# D3571) for 5 minutes at room temperature in the dark.

To measure the compound-induced inhibition of viral surface antigen expression, a cell-based immunostaining assay was performed as previously described [21 (link)], with minor modifications. Briefly, Vero cells (ATCC CCL-81) were seeded onto 96-well microtitration plates and 24 h later the confluent monolayers were infected with SARS-CoV-2 at MOI of 0.1. Then, diphyllin or cleistantin B were added at different concentrations to cell monolayers that were then cultured for 48 h at 37 °C. After cold acetone–methanol (1:1, v/v) fixation and blocking with 10% fetal bovine serum, cells were incubated with a rabbit (2019-nCoV) spike S1 antibody (1:50; Sino Biological, Duesseldorfer, Germany) and subsequently incubated for 1 h at 37 °C with anti-rabbit goat secondary antibodies conjugated with fluorescein isothiocyanate (FITC; 1:250; Sigma–Aldrich, Prague, Czech Republic). Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (1 μg/mL) for the visualization of cell nuclei and fluorescence signals were recorded with an Olympus IX71 epifluorescence microscope.