Nicolas Schrantz, Ph.D.
Nicolas Schrantz, Ph.D., a Senior Manager of R&D at Thermo Fisher Scientific, leads a team of R&D scientists responsible for the development of a novel arginase antibody for studying macrophages by flow cytometry. His team is involved with the development of new Invitrogen™ eBioscience™ antibodies for flow cytometry. He speaks to us about the anti-human/mouse Arginase 1 antibody development and shares insights into the performance and tips for use of antibody conjugates in multiplex flow cytometry applications.
Please tell me about the Arginase antigen.
Arginase 1 (Arg1) is a 35 kDa enzyme that converts L-arginine to urea and L-ornithine, which is the final step in the urea cycle. The resulting polyamines are important for cell proliferation and removal of toxins that arise from protein degradation. By degrading arginine, Arginase 1 deprives NO synthase of its substrate and down-regulates nitric oxide production.
In both human and mouse, Arginase 1 is expressed in the liver, neutrophils, myeloid derived suppressor cells (MDSC), and neural stem cells. In human, expression in blood neutrophils but not in CCR3+ (CD193) granulocytes has been reported.1,2,3 In mice, expression of Arginase 1 is one of the hallmarks of alternatively activated macrophages (M2a). Arginase 1 may be expressed in the myeloid cells infiltrating tumors, and is typically found in the majority of hepatocellular carcinomas. Defects in Arginase 1 are the cause of argininemia, an autosomal recessive disorder characterized by hyperammonemia.1,2,3
The monoclonal antibody A1exF5 recognizes both human and mouse Arginase 1, a cytosolic enzyme (Arg1). This A1exF5 clone is compatible with both the standard intracellular protocols (Figure 1) and the Foxp3/Transcription Factor Staining Buffer Set (Cat No. 00-5523-00, data not shown). This clone is the only one available that can be used to detect by flow cytometry human or mouse Arginase 1.
Figure 1. Expression of Arginase 1 in human blood neutrophils.
Normal human lysed whole blood cells were stained intracellularly, using the Intracellular Fixation & Permeabilization Buffer Set (Cat. No. 88-8824-00) and protocol, with 0.5 µg Arginase 1 Monoclonal Antibody, APC (Cat. No. 17-3697). Cells were co-stained with CD16 Monoclonal Antibody, FITC (Cat. No. 11-0168-42) and CD193 (CCR3) Monoclonal Antibody, PE (Cat. No. 12-1939-42). Cells in the granulocyte gate (left panel) were used for analysis and were gated on either the CD193+ population (blue histogram) or the CD16+ population (purple histogram).
Figure 2. Expression of Arginase 1 in mouse bone marrow derived M2a macrophages.
Intracellular staining of mouse bone marrow derived macrophages polarized to M1 or M2a. As expected based on known relative expression patterns, Arginase 1 clone A1exF5 stains most M2a macrophages (right panel) and does not stain M1 macrophages (left panel). Details: C57BL/6 mouse bone marrow derived macrophages were polarized for 24 hours with either IL-4 or LPS and IFN-gamma, and subsequently surface stained with F4/80 (clone BM8). Cells were then fixed and permeabilized with the IC Fixation & Permeabilization Buffer Set (Cat. No. 88-8824-00) and protocol followed by intracellular staining with Arginase 1 (clone A1exF5). Total viable cells were used for analysis as determined by Fixable Viability Dye.
What type of research can utilize Arginase 1?
Arginase 1 is a key effector and marker of M2a macrophages and myeloid derived suppressor cells (MDSC) that are major mediators of T cells suppression. This marker is particularly important for researchers studying myeloid cells, inflammation, and tumor microenvironment. It is present in both humans and in mice.
If incorporating Arginase 1 into a flow cytometry panel, what other markers would be recommended for use and why?
Our monoclonal antibody A1exF5 recognizes both human and mouse Arginase 1 and can be added to any panel aiming to identify differentiated macrophages and their activation state. Depending on the studied species, A1exF5 can be used with the below non-exhaustive list of markers (Table 1).
Table 1. Suggested markers to use with the Invitrogen™ eBioscience™ Arginase 1 monoclonal antibody (clone A1exF5).
During the development of A1exF5, our validation process lead us to an important discovery. We studied in depth the expression of Arginase 1 and other previously developed markers on mouse Large Peritoneal Macrophages and found that these cells can be further subdivided based on the expression specific markers.
Peritoneal macrophages comprise two distinct populations: Small Peritoneal Macrophages (SPM), also known as CD11b (med/high) or F4/80 (low) macrophages, and Large Peritoneal Macrophages (LPM), also known as CD11b (high) or F4/80 (high) macrophages. The SPM cells are monocyte-derived whereas the LPM have characteristics of tissue macrophages. The LPM were considered a relatively homogenous population but our data indicates that this population can be further subdivided based on the expression of Arginase 1 and VSIG4. The LPM can be further characterized by their expression of CD206 and CD163. Our data shows that the distribution of these subpopulations in the three tested mouse strains significantly varies. The relationship between these phenotypes and functional properties of these cells is yet to be unraveled.
Figure 3: Expression of Arginase 1, VISIG4, CD206, and CD163 on large peritoneal macrophages from different mouse strains
Resident peritoneal exudate from the indicated mouse strains were Fc blocked using mouse serum and CD16/CD32 Monoclonal Antibody (Cat. No. 14-0161-86), and surface-stained with: F4/80 eFluor 450 (Cat. No. 48-4801-82), CD11b FITC (Cat. No. 11-0112-82), VSIG4 PE-Cyanine7 (Cat. No. 25-5752-82), and CD163 PerCP-eF710 (Cat. No. 46-1631-82). The cells were then fixed and permeabilized with the IC Fixation & Permeabilization Buffer Set (Cat. No. 88-8824-00) and intracellularly stained with: CD206 PE (Cat No. 12-2061-82) and Arginase 1, and analyzed by Flow Cytometry.
What can you tell me about the relative expression of Arginase 1?
The expression of Arginase 1 in human or mouse activated macrophages is medium to high.
Any tips and tricks you recommend for using Arginase 1 in flow cytometry panels?
Fixation of this antigen works the best with Intracellular Fixation & Permeabilization Buffer set (Cat. No. 88-8824-00) but it is also compatible with eBioscience™ Foxp3/Transcription Factor Staining Buffer Set (Cat. No. 00-5523-00).
Is the Arginase 1 A1exF5 clone available for use in other applications?
The A1exF5 clone has not been tested for applications other than flow cytometry, but we developed and validated the clone sl6Arg for immunocytochemistry (ICC), immunofluorescence (IF) and western blot, and 2 additional clones are used in our Luminex assay, Arginase 1 Human ProcartaPlex™ Simplex Kit (Cat. No. EPX010-12216-901).
What key references should I review if I want to learn more about Arginase 1?
- Human eosinophil granulocytes do not express the enzyme arginase. J Leukoc Biol. 2010 Jun;87(6):1125-32.
- Arginase 1 expressed in myelocytes/ metamyelocytes and localized in gelatinase granules of human neutrophils. Blood 2007 109:3084-3087.
- “Of Mice and Men”: Arginine Metabolism in Macrophages. Front Immunol. 2014; 5: 479.
Arginase 1 Monoclonal Antibodies
|Arginase 1 Monoclonal Antibody (A1exF5), PE-Cyanine7, eBioscience™||25-3697|
|Arginase 1 Monoclonal Antibody (A1exF5), eFluor 450, eBioscience™||48-3697|
|Arginase 1 Monoclonal Antibody (A1exF5), Alexa Fluor 488, eBioscience™||53-3697|
|Arginase 1 Monoclonal Antibody (A1exF5), APC, eBioscience™||17-3697|
|Arginase 1 Monoclonal Antibody (A1exF5), PE, eBioscience™||12-3697|
|Arginase 1 Monoclonal Antibody (A1exF5), Alexa Fluor 700, eBioscience™||56-3697|
|Arginase 1 Monoclonal Antibody (A1exF5), PerCP-eFluor 710, eBioscience™||46-3697|
Search the entire catalog for all currently available Arginase 1 (clone A1exF5) Monoclonal Antibodies.
Interested in reading additional Behind the Bench blog posts for Flow Cytometry? See blog posts for flow cytometry.
For Research Use Only. Not for Use in Diagnostic Procedures.