Nicolas Schrantz, Ph.D.
Nicolas, a Senior Manager of R&D at Thermo Fisher Scientific, leads a team of R&D scientists responsible for the development of a mCD163 antibody for studying macrophages by flow cytometry. He speaks to us about the anti-mouse CD163 antibody (clone TNKUPJ) development and shares insights into the performance and tips for use of antibody conjugates in multiplex flow cytometry applications.
Please tell me about the CD163 antigen.
CD163 is a 130kDa surface receptor expressed by certain subsets of tissue macrophages, including splenic red pulp macrophages, Kupffer cells, intestinal lamina propria macrophages and a small fraction of peritoneal macrophages. In contrast to human blood monocytes, mouse monocytes do not express CD163. Also, unlike human CD163, mouse CD163 is not as readily induced by M2 polarizing cytokines, and it is not a good marker of M2 macrophages. No common cell lines of monocytic or macrophage origin express mouse CD163. Unlike in mouse, in Human CD163 has been shown to be proteolytically cleaved from the cell surface monocyte and able to act as a soluble anti-inflammatory factor.
What type of research can utilize anti-mouse CD163, clone TNKUPJ?
CD163 is a macrophage scavenger receptor mainly known for its capacity to bind and internalize haptoglobin-hemoglobin complexes. CD163 is also a receptor for TNF-related weak inducer of apoptosis (TWEAK), an erythroblast adhesion molecule, and a receptor for different bacteria and viruses. This TNKUPJ antibody is particularly important for researchers studying myeloid cells, inflammation, tumor microenvironment, angiogenesis, sepsis and atherosclerosis.
If incorporating anti-mouse CD163, clone TNKUPJ into a flow cytometry panel, what other markers would be recommended for use and why?
Our monoclonal antibody TNKUPJ recognizes mouse CD163 and can be added to any panel aiming to characterize macrophages. This TNKUPJ antibody can be used with the below non-exclusive list of mouse markers (Table 1).
Table 1. Non-exhaustive list of markers that can be used with TNKUPJ.
During the development of TNKUPJ, we studied the expression of mouse CD163 on splenic and peritoneal macrophages in depth. In Balb/c mice, CD163 is expressed by almost half of the splenic F4/80 expressing cells, where in the peritoneal cavity it is only expressed by a small fraction of the F4/80 expressing cells.
Figure 1. CD163 expression in macrophages. Balb/c splenocytes and resident peritoneal exudate cells were Fc blocked, stained with anti-mouse CD163 (clone TNKUPJ), and co-stained with anti-mouse F4/80 eFluor 450 (clone BM8) and anti-mouse CD11b APC (clone M1/70). Although nearly a half of spleen F4/80+ macrophages were CD163 positive, only a small fraction of small peritoneal macrophages (SPM) and large peritoneal macrophages (LPM) appeared to express CD163. Peritoneal lymphocytes (B, T), mast cells (MC) and eosinophils (Eo) were CD163 negative.
What can you tell me about the relative expression of anti-mouse CD163, clone TNKUPJ?
The expression of anti-mouse CD163 in mouse is medium to high compared to other bright markers like CD4 or CD8.
Any tips and tricks you recommend for using anti-mouse CD163, clone TNKUPJ in flow cytometry panels?
This TNKUPJ antibody will detect CD163 on fixed and permeabilized cells (Intracellular Fixation & Permeabilization Buffer set), allowing for staining of the intracellular pool of this receptor. Although CD163 is relatively stable to collagenase digestion, aggressive tissue dissociation protocols might potentially decrease the amount of surface CD163. In these cases, intracellular detection is recommended.
Is the anti-mouse CD163, clone TNKUPJ available for use in other applications?
The TNKUPJ clone has also been validated for Immunohistochemistry on fixed, frozen mouse tissue sections (spleen and liver).
Figure 2. Immunohistochemistry of mouse spleen. Frozen mouse spleen was stained with 5 µg/mL Rat IgG2a kappa Isotype Control (left) or anti-mouse CD163 (right) followed by anti-mouse IgG TRITC. Nuclei are stained with DAPI.
Figure 3. Immunohistochemistry of mouse liver. Frozen mouse liver stained with 5 µg/mL Rat IgG2a kappa Isotype Control (left) or anti-mouse CD163 (right) followed by anti-mouse IgG TRITC. Nuclei are stained with DAPI.
What key references should I review if I want to learn more about anti-mouse CD163?
Identification of CD163 as an anti-inflammatory receptor for HMGB1-haptoglobin complexes. JCI Insight. 2016;1(7):e85375.
CD163 and inflammation: biological, diagnostic, and therapeutic aspects. Antioxid Redox Signal. 2013 Jun 10;18(17):2352-63.
CD163+ macrophages promote angiogenesis and vascular permeability accompanied by inflammation in atherosclerosis. J Clin Invest. 2018 Mar 1;128(3):1106-1124.
CD163 and IgG codefend against cytotoxic hemoglobin via autocrine and paracrine mechanisms. J Immunol. 2013 May 15;190(10):5267-78.
Molecular cloning and characterization of the mouse CD163 homologue, a highly glucocorticoid-inducible member of the scavenger receptor cysteine-rich family. Immunogenetics. 2001 Mar;53(2):170-7.
Human eosinophil granulocytes do not express the enzyme arginase. J Leukoc Biol. 2010 Jun;87(6):1125-32.
Table 2. Anti-mouse CD163 (TNKUPJ) monoclonal antibodies.
|CD163 Monoclonal Antibody (TNKUPJ), eBioscience™||14-1631-82|
|CD163 Monoclonal Antibody (TNKUPJ), PerCP-eFluor 710, eBioscience™||46-1631-82|
|CD163 Monoclonal Antibody (TNKUPJ), PE, eBioscience™||12-1631-82|
Search the entire catalog for all available CD163 (clone TNKUPJ) monoclonal antibodies.
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