Fig. 2 Detection of SARS-CoV-2 RBD binding to human ACE2 receptor. a Flow cytometry analysis of receptor expression in stable cell lines. (left panel) 293T cells alone expressed neither human ACE2 (hACE2) receptor (orange line) nor hDPP4 receptor (cyan line); (middle panel) hACE2-expressing 293T (hACE2/293T) cells expressed only hACE2 (orange line), but not hDPP4 (cyan line); (right panel) hDPP4-expressing 293T (hDPP4/293T) cells expressed only hDPP4 (cyan line), but not hACE2 (orange line). Mock-incubated cells (gray shading) were used as control. Representative images and median fluorescence intensity (MFI) ± standard error (s.e.m.) were shown (n = 4). b, c Flow cytometry analysis of SARS-CoV-2 RBD binding to cell-associated hACE2 receptor in hACE2/293T stable cell lines. SARS-CoV-2 RBD protein bound strongly to hACE2/293T cells (b (left panel, red line)), but not to hDPP4/293T cells (c (left panel, violet line)). SARS-CoV RBD protein bound to hACE2/293T cells (b (middle panel, red line)), but not to hDPP4/293T cells (c (middle panel, violet line)). MERS-CoV RBD protein did not bind to hACE2/293T cells (b (right panel, red line)), but rather bound to hDPP4/293T cells (c (right panel, violet line)). Human IgG Fc (hIgG-Fc, hereinafter hFc) protein-incubated cells (blue line) and mock-incubated cells (gray shading) were included as controls (b, c). Representative images and MFI ± s.e.m. were shown (n = 4). d Immunofluorescence detection of SARS-CoV-2 RBD binding to cell-associated hACE2 receptor in hACE2/293T cells. SARS-CoV-2 RBD protein (green) and SARS-CoV RBD protein (green), each of which was fused with a C-terminal hFc, were stained with FITC-labeled goat anti-human IgG antibody (1:500). hACE2 was stained with a goat-anti-hACE2 antibody (5 μg/ml) and Alexa-Fluor 647-labeled anti-goat antibody (red) (1:200). Fc-fused MERS-CoV RBD protein did not bind to hACE2, so only hACE2 (red), but not RBD (green), was detected in hACE2/293T cells. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, blue). Scale bar: 10 μm. Representative images are shown. e Detection of dose-dependent binding of SARS-CoV-2 RBD protein to soluble hACE2 (sACE2) receptor by ELISA. The SARS-CoV-2 RBD binding to soluble hDPP4 (sDPP4) receptor (f), and the binding of both SARS-CoV RBD and MERS-CoV RBD proteins to sACE2 (e), or sDPP4 (f), were tested. Control: hFc protein. Data are presented as mean A450 ± s.e.m. (n = 4). 50% effective dose (EC50) was calculated for the binding between SARS-CoV-2 RBD (black) or SARS-CoV RBD (red) and hACE2 protein (e, sACE2), or the binding between MERS-CoV RBD and hDPP4 protein (sDPP4, green) (f). g–i Flow cytometry analysis of inhibition of SARS-CoV-2 RBD protein binding to hACE2/293T cells by sACE2. Binding of SARS-CoV-2 RBD to hACE2/293T cells (g, h, green line) was blocked by sACE2 (g, black line), but not by sDPP4 (h, red line). hFc protein-incubated cells (blue line) and mock-incubated cells (gray shading) were included as controls (g, h). Representative images are shown. i The blocking ability of sACE2 or sDPP4, as described above, was expressed as MFI ± s.e.m. (n = 4). Low MFI correlates with high blockage. Experiments were repeated twice and yielded similar results