anti ace2  (R&D Systems)

Journal: PLOS Pathogens

Article Title: B Cell Receptor’s function in virus entry: Anti-SARS-CoV-2 B cell receptors can mediate viral entry in an ACE2-independent mechanism

doi: 10.1371/journal.ppat.1013946

Figure Lengend Snippet: A) Binding of Scalet-labeled lentiviral vector pseudotyped with S protein to 293T cells with or without ectopic expression of TIM-1, ACE2, as well as CD79 293T cells expressing control (anti-HIV-1 3BNC117), or anti-SARS-CoV-2 BCR (REGN10933 and CB6) at multiple MOIs (µg p24/ml). The cells were incubated with the vector for 1 hour at 37°C, and Scarlet-positive populations were quantified as a measure of virus binding via flow cytometry. B) Fusion/entry of β-lactamase containing lentiviral vector pseudotyped with S protein with or without ectopic expression of TIM-1, ACE2, control, or anti-SARS-CoV-2 BCR. The cells and virus were incubated for 1 hour at 37°C. The cells were then incubated with CCF4-AM for 1 hour at RT. Using flow cytometry, entry of virus is quantitated by cleavage of CCF4-AM in the cytoplasm of target cells. The positive signal in the BV421 channel was measured as virus fusion. N = 3 and averages are shown with SD (error bar). This experiment was repeated twice in singlicate and once in triplicate, and the results of the triplicated experiments are shown. C) Transduction of 293T cells with or without ectopic expression of TIM-1, ACE2, as well as CD79 293T cells with ectopic expression of control, or anti-SARS-CoV-2 BCR with lentiviral vector pseudotyped with S protein, carrying an EGFP transgene. Three days after transduction, transduction was quantified by analyzing EGFP transgene expression using flow cytometry. This experiment was repeated three times in singlicate and once in triplicate, and the results of the triplicated experiments are shown. D) Transduction of CD79 Sp2/0 cells with or without ectopic expression of ACE2, control or anti-SARS-CoV-2 BCR with lentiviral vector pseudotyped with S protein carrying an EGFP transgene in the absence and or presence of reverse transcriptase inhibitor (RTI), (Nevirapine, 10 µM). Three days after transduction, transduction was quantified by analyzing EGFP transgene expression using flow cytometry. N = 3 and averages are shown with SD (error bar). This experiment was repeated twice in singlicate and once in triplicate, and the results of the triplicated experiments are shown.

Article Snippet: 293T, ACE2 293T, CD79 293T, REGN CD79 293T, and CB6 CD79 293T cells (5 × 10 5 cells) were incubated with either a control mouse IgG1 antibody (10 μg/ml) (Biolegend), an anti-human ACE2 antibody (AdipoGen, San Diego, CA), or E64d (20 μM)(Selleckchem, Austin, TX) at 37°C for 20 minutes, followed by infection with a 5-fold dilution of SARS-CoV-2 VLP.

Techniques: Binding Assay, Labeling, Plasmid Preparation, Expressing, Control, Incubation, Virus, Flow Cytometry, Transduction, Reverse Transcription

Journal: PLOS Pathogens

Article Title: B Cell Receptor’s function in virus entry: Anti-SARS-CoV-2 B cell receptors can mediate viral entry in an ACE2-independent mechanism

doi: 10.1371/journal.ppat.1013946

Figure Lengend Snippet: A) 293T cells, with or without ectopic expression of ACE2, were infected with SARS-CoV-2 VLPs carrying a firefly luciferase reporter gene. These infections were performed in the presence or absence of control antibodies (Block A), anti-ACE2 antibodies (Block B), or the protease inhibitor E64d (Block C). Two days post-infection, viral infection was quantified by measuring firefly luciferase activity in the lysates of the infected cells. N = 3 and averages are shown with SD (error bar). This experiment was repeated twice in singlicate and once in triplicate, and the results of the triplicated experiments are shown. B) Infection of CD79 293T cells, with or without ectopic expression of anti-SARS-CoV-2 BCR (REGN10933 and CB6), with SARS-CoV-2 VLP carrying a firefly luciferase reporter gene in the presence and absence of control (Block A) and anti-ACE2 antibodies (Block B), or E64d (Block C). N = 3 and averages are shown with SD (error bar). Two days post-infection, viral infection was quantified by measuring firefly luciferase activity in the lysates of the infected cells. This experiment was repeated twice in singlicate and once in triplicate, and the results of the triplicated experiments are shown. C) CD79 Sp2/0 cells, with or without ectopic expression of ACE2, a control BCR, or an anti-SARS-CoV-2 BCR (ScFv form), were infected with either live or heat-inactivated SARS-CoV-2. Two days after infection, the RNA was isolated from infected cells and the copy numbers of mouse GAPDH and SARS-CoV-2 N1 and N2 RNA copy numbers were quantified by digital droplet PCR. The copy numbers of SARS-CoV-2 N1 (left panel) or N2 (right panel) per 100 copies of GAPDH are shown with SD (error bar). N = 3. Significance was calculated using a two-sample two-sided unpaired student t-test (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001). D) Supernatants from SARS-CoV-2 -infected cells (shown in panel C) were harvested at 48 hours post-infection and used to inoculate Vero E6 cells. Two days after infection, the RNA was isolated from infected Vero E6 cells and the copy numbers of RRP30 and SARS-CoV-2 N1 and N2 RNA copy numbers were quantified by digital droplet PCR. The copy numbers of SARS-CoV-2 N1 (left panel) or N2 (right panel) per 1000 copies of RRP30 are shown with SD (error bar). N = 3. For panels A and B, significance was calculated using a two-sample two-sided unpaired student t-test (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: 293T, ACE2 293T, CD79 293T, REGN CD79 293T, and CB6 CD79 293T cells (5 × 10 5 cells) were incubated with either a control mouse IgG1 antibody (10 μg/ml) (Biolegend), an anti-human ACE2 antibody (AdipoGen, San Diego, CA), or E64d (20 μM)(Selleckchem, Austin, TX) at 37°C for 20 minutes, followed by infection with a 5-fold dilution of SARS-CoV-2 VLP.

Techniques: Expressing, Infection, Luciferase, Control, Blocking Assay, Protease Inhibitor, Activity Assay, Isolation