Flow Cytometry Workflows for COVID-19 Research

Anti-ACE-2 and other flow cytometry antibodies from R&D Systems for SARS-CoV-2 and COVID-19 research.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was identified as the causative agent of the respiratory disease, COVID-19. COVID-19 has developed into a global pandemic, thus there is a pressing urgency to understand the pathogenesis of SARS-CoV-2. It has been shown that SARS-CoV-2 uses Angiotensin-Converting Enzyme 2 (ACE-2), a type I integral membrane protein, as a cellular entry receptor. The Spike (S) protein on SARS-CoV-2 binds to ACE-2 through its receptor binding domain (RBD). Binding of the S protein to ACE-2 is the first step of viral entry into host cells.

 

ACE-2 Detection by Flow Cytometry

ACE-2 is expressed throughout the body, however, the cell subsets targeted by SARS-CoV-2 is still not completely known. Additionally, researchers are still discovering which factors regulate ACE-2 expression. R&D Systems offers monoclonal and polyclonal antibodies conjugated to a variety of fluorochromes for investigating ACE-2 expression by flow cytometry.

R&D Systems™ Anti-ACE-2 Flow Cytometry Antibodies
Clone Unconjugated Antibody (Catalog #) Fluorochrome-Conjugated Antibodies (Catalog # - Fluorochrome)
535919 MAB9332 FAB9332-G, N, R, S, T, U, V
Polyclonal AF933 FAB933-G, N, R, S, T, U, V

Fluorochrome Key:
G Alexa Fluor® 488, N Alexa Fluor 700, R Alexa Fluor 647, S Alexa Fluor 750, T Alexa Fluor 594, U Alexa Fluor 350, V Alexa Fluor 405

Detection of ACE-2 in the HEK293 Human Cell Line Transfected with Human ACE-2 and eGFP by Flow Cytometry. The HEK293 human embryonic kidney cell line transfected with (A) human ACE-2 or (B) an irrelevant protein, and eGFP. The cells were then stained with a Mouse Anti-Human Alexa Fluor 647-Conjugated ACE-2 Monoclonal Antibody (R&D Systems, Catalog # FAB9332R). Quadrant markers were set based on Alexa Fluor 647-Conjugated Mouse IgG2A Isotype Control (R&D Systems, IC003R, data not shown). Staining was performed using our Staining Membrane-Associated Proteins protocol.

Flow cytometry analysis of ACE-2 expression using R&D Systems™ Mouse Anti-Human Alexa Fluor® 647-Conjugated ACE-2 Monoclonal Antibody.

 

Detection of ACE-2 in HEK293 Human Cell Line transfected with Human ACE-2 and eGFP by Flow Cytometry. HEK293 human embryonic kidney cell line transfected with (A) human ACE-2 or (B) an irrelevant protein, and eGFP. The cells were then stained with a Goat Anti-Human ACE-2 Alexa Fluor 700-Conjugated Affinity-Purified Polyclonal Antibody (R&D Systems, Catalog # FAB933N). Staining was performed using our Staining Membrane-Associate Proteins protocol.

Flow cytometry analysis of ACE-2 expression using R&D Systems™ Goat Anti-Human ACE-2 Alexa Fluor® 700-Conjugated Affinity-Purified Polyclonal Antibody.

 

Flow Cytometry Blocking Assays

Receptor binding is the first step in viral infection. For coronaviruses, a cell-surface S protein mediates their entry into host cells. The S protein on SARS-CoV-2 recognizes and binds to ACE-2 through its RBD. Thus, both proteins could be viable therapeutic targets. Blocking the interaction between ACE-2 and the S protein, for example with antibodies directed against either ACE-2 or the S Protein RBD, could, in theory, prevent SARS-CoV-2 infection. The development of COVID-19 therapeutics, though, is encumbered by the fact that SARS-CoV-2 exhibits high pathogenicity and infectivity and needs to be handled under biosafety level 3 conditions. To propel the development of COVID-19 therapeutics, we developed a flow cytometry-based, in vitro assay for evaluating the effectiveness of antibodies and/or small molecules to block the binding of the S protein to ACE-2.

An ACE-2 blocking antibody blocks SARS-CoV-2 binding to ACE-2 expressed on a transfected cell line.
ACE-2 Flow Cytometry Blocking Assay Principle. Cells are transfected with human ACE-2 DNA and express the receptor on their cell surface. When incubated with a recombinant His-tag SARS-CoV-2 Protein RBD, the protein will bind to ACE-2 (A). SARS-CoV-2 binding is detected with an APC-Conjugated His-Tag Antibody. Incubating the cells with an ACE-2 blocking antibody prior to the SARS-CoV-2 Protein RBD allows the ACE-2 antibody to bind to the ACE-2 receptor, blocking SARS-CoV-2 binding (B). R&D Systems also offers a SARS-CoV-1/2 Spike RBD Llamabody™ Antibody that binds to the S Protein RBD and blocks it from binding to ACE-2.

 

Flow Cytometry Blocking Assay Tools
Product Brand Catalog # Additional Information
Recombinant SARS-CoV-2 S Protein RBD R&D Systems 10499-CV HEK293-Derived, FC-Tagged
Recombinant SARS-CoV-2 S Protein RBD R&D Systems 10500-CV HEK293-Derived, His-Tagged
Recombinant SARS-CoV-2 S Protein RBD R&D Systems 10534-CV CHO-Derived, His-Tagged
Recombinant SARS-CoV-2 S Protein RBD R&D Systems 10523-CV Tn5-Derived, His-Tagged
Recombinant SARS-CoV-2 S Protein RBD Novus Biologicals NBP2-90982 His-Tagged
Goat Anti-Human ACE-2 Polyclonal Antibody R&D Systems AF933 Conjugates Available
Mouse Anti-Human ACE-2 Monoclonal Antibody Novus Biologicals NBP2-80038 Conjugates Available
SARS-CoV-1/2 Spike Llamabody Antibody R&D Systems LMAB10541 New camelid antibody

Blocking SARS-CoV-2 Binding to ACE-2 with an Anti-ACE-2 Antibody. In a functional flow cytometry test, sheep Fc-tagged recombinant human Spike S1 binds to HEK293 human embryonic kidney cell line transfected with human ACE-2 (black dotted line). Protein binding was detected with a Donkey Anti-Sheep APC-Conjugated IgG Secondary Antibody (R&D Systems, Catalog # F0127). S1 binding to ACE-2 is completely blocked by a Mouse Anti-Human ACE-2 Monoclonal Antibody (Novus Biologicals, Catalog # NBP2-80038; orange histogram). A Mouse Anti-Human DC-SIGN/CD209 Monoclonal Antibody (R&D Systems, Catalog # MAB161) was used as an irrelevant control (blue line).

Detection of ACE-2 in the presence and absence of an ACE-2 blocking antibody by a fluorescent-tagged Recombinant SARS-CoV-2 Spike Protein RBD.

 

Blocking SARS-CoV-2 Binding to ACE-2 With a SARS-CoV-1/2 Spike RBD Llamabody. In a functional flow cytometry test, a His-Tagged Recombinant SARS-CoV-2 Spike RBD Protein (R&D Systems, 10534-CV) binds to HEK293 human embryonic kidney cell line transfected with human ACE-2 (black dotted line). Protein binding was detected with an APC-Conjugated Mouse Anti-His Tag Monoclonal Antibody (R&D Systems, Catalog # IC050A). Spike RBD binding to ACE-2 is completely blocked by an Anti-SARS CoV-1/2 Spike RBD Llamabody Antibody (R&D Systems, Catalog # LMAB10541; orange histogram). A Mouse Anti-Human DC-SIGN/CD209 Monoclonal Antibody (R&D Systems, Catalog # MAB161) was used as an irrelevant control (blue line).

Detection of ACE-2 in the presence and absence of a blocking SARS-CoV-1/2 S Protein RBD Llamabody.

 

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Monitoring Immune Cell Responses

For some individuals, infection with SARS-CoV-2 can result in the development of moderate to severe acute respiratory distress syndrome (ARDS). One of the main features of ARDS is the cytokine storm, which is an uncontrolled systemic inflammatory response resulting from the excessive production of proinflammatory cytokines and chemokines. Clinical investigations of patients severely ill with COVID-19 have shown that these individuals display cytokine storm symptoms. Despite these findings, the immune response to SARS-CoV-2 infection is still only partially understood.

We offer an extensive selection of antibodies validated for flow cytometry that allow you to detect and analyze specific cell surface and intracellular markers in order to identify and characterize different immune cell types and cell type-specific subsets activated in response to SARS-CoV-2 infection. Browse R&D Systems and Novus Biologicals™ selection of flow cytometry antibodies. Or explore our Interactive Cell Marker Tool to find cell surface and intracellular markers used to identify different immune cell types. Markers link directly to R&D Systems and Novus Biologicals products, making it easy to find antibodies. Additionally, Bio-Techne offers workflow solutions for investigating the role of different immune cells, such as monocytes, macrophages, dendritic cells, natural killer cells, T cells and B cells, in the immunopathogenesis of COVID-19.

Visit Bio-Techne's Immunology Workflow Solutions site to view products for:

  • Immune Cell Selection and Culture
  • Immune Cell Identification and Characterization
  • Modulation of Immune Cells
  • Investigation and Quantification of Immune Responses