2.10 SARS-CoV-2 Spike-ACE2 binding assay
A recombinant protein containing receptor-binding domain (RBD) of the
SARS-CoV2 spike protein fused with GFP (S-RBD-GFP) were produced in
Expi293F cells after transfection of pcDNA3-SARS-CoV-2-S-RBD-sfGFP (a
gift from Erik Procko, Addgene plasmid # 141184) using the
ExpiFectamine™ 293 transfection kit and grown in Expi293 expression
medium (Thermo Fisher Scientific, USA) for 4-5 days. The culture medium
was cleared via centrifugation at 800×g for 5 min and stored at 4 °C
until analysis. For the binding assay, ACE2+ H1299
cells were treated with DMSO or indicated concentrations of CL extracts
or LA for 30 min, followed by incubation with a 1/10 dilution of medium
containing S-RBD-GFP for 10 min at 37°C in CO2 incubator.
ACE2+ H1299 cells were then washed with PBS containing
1% BSA and analyzed for the binding of S-RBD-GFP using LSRFortessa flow
cytometer (BD Biosciences). The data was analyzed using FlowJo software
(BD Life Sciences).