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class="content-sidebar-wrap"><main class="content"><article class="post-11626 post type-post status-publish format-standard has-post-thumbnail category-general category-immunology tag-flow-cytometry tag-immunology entry" aria-label="Anti-mouse CD163: A macrophage marker for flow cytometry"><header class="entry-header"><h1 class="entry-title">Anti-mouse CD163: A macrophage marker for flow cytometry</h1> <span class="post-info-author"> By <a href="https://www.thermofisher.com/blog/author/suzannebuck/" title="Posts by Suzanne Buck, PhD" rel="author">Suzanne Buck, PhD</a> </span> <span class="post-info-date"> 09.26.2018 </span> </header><div class="entry-content"><p><strong><img decoding="async" class="alignnone" src="https://www.thermofisher.com/blog/wp-content/uploads/sites/9/2018/09/arginase-nicolas-schrantz.jpg" alt="headshot" width="150" height="198" />Nicolas Schrantz, Ph.D.</strong><br /> <span style="font-size: medium">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.</span></p> <h2>Please tell me about the CD163 antigen.</h2> <p><span style="font-size: medium">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.</span></p> <h2>What type of research can utilize anti-mouse CD163, clone TNKUPJ?</h2> <p><span style="font-size: medium">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 <a href="https://www.thermofisher.com/antibody/primary/query/TNKUPJ?icid=BID_FCREG_PJT3273_TNKUPJ-antibody">TNKUPJ antibody</a> is particularly important for researchers studying myeloid cells, inflammation, tumor microenvironment, angiogenesis, sepsis and atherosclerosis.</span></p> <h2>If incorporating anti-mouse CD163, clone TNKUPJ into a flow cytometry panel, what other markers would be recommended for use and why?</h2> <p><span style="font-size: medium">Our <a href="https://www.thermofisher.com/antibody/primary/query/TNKUPJ?icid=BID_FCREG_PJT3273_monoclonal-antibody-TNKUPJ">monoclonal antibody TNKUPJ</a> 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).</span></p> <p><strong>Table 1. Non-exhaustive list of markers that can be used with TNKUPJ.</strong><img decoding="async" class=" wp-image-11636 alignnone" src="https://www.thermofisher.com/blog/wp-content/uploads/sites/9/2018/09/TNKUPJ-table-1.png" alt="" width="700" height="306" srcset="https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2018/09/TNKUPJ-table-1.png 847w, https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2018/09/TNKUPJ-table-1-300x131.png 300w, https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2018/09/TNKUPJ-table-1-768x335.png 768w" sizes="(max-width: 700px) 100vw, 700px" /><br /> <span style="font-size: medium">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.</span></p> <p><img loading="lazy" decoding="async" class="aligncenter wp-image-11900 size-full" src="https://www.thermofisher.com/blog/wp-content/uploads/sites/9/2018/09/Fig1-CD163-expression-in-macrophages.jpg" alt="10 panels total, all dot plots. First 3 plots show gating in spleen sample, final plot is CD163 PE vs. F4/80 eFluor 450. Remaining 7 plots show gating in the resident peritoneal exudate cells (first 3 plots), and 4 plots show CD163 PE staining in different cell subsets. " width="500" height="470" srcset="https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2018/09/Fig1-CD163-expression-in-macrophages.jpg 500w, https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2018/09/Fig1-CD163-expression-in-macrophages-300x282.jpg 300w" sizes="(max-width: 500px) 100vw, 500px" /><br /> <strong>Figure 1. CD163 expression in macrophages. </strong>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.</p> <h2>What can you tell me about the relative expression of anti-mouse CD163, clone TNKUPJ?</h2> <p><span style="font-size: medium">The expression of anti-mouse CD163 in mouse is medium to high compared to other bright markers like CD4 or CD8.</span></p> <h2>Any tips and tricks you recommend for using anti-mouse CD163, clone TNKUPJ in flow cytometry panels?</h2> <p><span style="font-size: medium">This TNKUPJ antibody will detect CD163 on fixed and permeabilized cells (<a href="https://www.thermofisher.com/order/catalog/product/88-8824-00?icid=BID_FCREG_PJT3273_intracellular-fixation">Intracellular Fixation & Permeabilization Buffer set</a>), 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.</span></p> <h2>Is the anti-mouse CD163, clone TNKUPJ available for use in other applications?</h2> <p><span style="font-size: medium">The TNKUPJ clone has also been validated for Immunohistochemistry on fixed, frozen mouse tissue sections (spleen and liver).</span></p> <p><img loading="lazy" decoding="async" class="aligncenter size-large wp-image-11901" src="https://www.thermofisher.com/blog/wp-content/uploads/sites/9/2018/09/Fig2-CD163-detection-in-mouse-spleen-via-IHC-1024x433.jpg" alt="2 panel microscopic image of staining with anti-mouse CD163." width="1024" height="433" /><br /> <strong>Figure 2. </strong><strong>Immunohistochemistry of mouse spleen.</strong> Frozen mouse spleen was stained with 5 µg/mL <a href="https://www.thermofisher.com/antibody/product/Rat-IgG2a-kappa-clone-eBR2a-Isotype-Control/14-4321-82?icid=BID_FCREG_PJT3273_rat-IgG2a">Rat IgG2a kappa Isotype Control</a> (left) or <a href="https://www.thermofisher.com/antibody/primary/query/TNKUPJ?icid=BID_FCREG_PJT3273_anti-mouse-CD163">anti-mouse CD163</a> (right) followed by <a href="https://www.thermofisher.com/antibody/product/Goat-anti-Rat-IgG-H-L-Secondary-Antibody-Polyclonal/26-4826-82?icid=BID_FCREG_PJT3273_anti-mouse-IgG">anti-mouse IgG TRITC</a>. Nuclei are stained with <a href="https://www.thermofisher.com/order/catalog/product/00-4959-52?icid=BID_FCREG_PJT3273_DAPI">DAPI</a>.</p> <p><img loading="lazy" decoding="async" class="aligncenter size-large wp-image-11902" src="https://www.thermofisher.com/blog/wp-content/uploads/sites/9/2018/09/Fig3-CD163-detection-in-mouse-liver-via-IHC-1024x445.jpg" alt="2 panel microscopic image of staining with anti-mouse CD163." width="1024" height="445" /><br /> <strong>Figure 3. </strong><strong>Immunohistochemistry of mouse liver.</strong> Frozen mouse liver stained with 5 µg/mL <a href="https://www.thermofisher.com/antibody/product/Rat-IgG2a-kappa-clone-eBR2a-Isotype-Control/14-4321-82?icid=BID_FCREG_PJT3273_Rat-IgG2a-2">Rat IgG2a kappa Isotype Control</a> (left) or <a href="https://www.thermofisher.com/antibody/primary/query/TNKUPJ?icid=BID_FCREG_PJT3273_Anti-Mouse-CD163-2">anti-mouse CD163</a> (right) followed by <a href="https://www.thermofisher.com/antibody/product/Goat-anti-Rat-IgG-H-L-Secondary-Antibody-Polyclonal/26-4826-82?icid=BID_FCREG_PJT3273_anti-mouse-IgG-2">anti-mouse IgG TRITC</a>. Nuclei are stained with <a href="https://www.thermofisher.com/order/catalog/product/00-4959-52?icid=BID_FCREG_PJT3273_DAPI-2">DAPI</a>.</p> <h2>What key references should I review if I want to learn more about anti-mouse CD163?</h2> <p><span style="font-size: medium">Identification of CD163 as an anti-inflammatory receptor for HMGB1-haptoglobin complexes. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4902170/">JCI Insight. 2016;1(7):e85375.</a></span></p> <p><span style="font-size: medium">CD163 and inflammation: biological, diagnostic, and therapeutic aspects. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3638564/">Antioxid Redox Signal. 2013 Jun 10;18(17):2352-63.</a></span></p> <p><span style="font-size: medium">CD163+ macrophages promote angiogenesis and vascular permeability accompanied by inflammation in atherosclerosis. <a href="https://www.ncbi.nlm.nih.gov/pubmed/29457790">J Clin Invest. 2018 Mar 1;128(3):1106-1124.</a></span></p> <p><span style="font-size: medium">CD163 and IgG codefend against cytotoxic hemoglobin via autocrine and paracrine mechanisms. <a href="https://www.ncbi.nlm.nih.gov/pubmed/23589619">J Immunol. 2013 May 15;190(10):5267-78.</a></span></p> <p><span style="font-size: medium">Molecular cloning and characterization of the mouse CD163 homologue, a highly glucocorticoid-inducible member of the scavenger receptor cysteine-rich family. <a href="https://www.ncbi.nlm.nih.gov/pubmed/11345593">Immunogenetics. 2001 Mar;53(2):170-7.</a></span></p> <p><span style="font-size: medium">Human eosinophil granulocytes do not express the enzyme arginase. <a href="https://www.ncbi.nlm.nih.gov/pubmed/20200399">J Leukoc Biol. 2010 Jun;87(6):1125-32.</a></span></p> <p><strong>Table 2. Anti-mouse CD163 (TNKUPJ) monoclonal antibodies.</strong></p> <table> <tbody> <tr> <td><strong>Product</strong></td> <td><strong>Cat. No.</strong></td> </tr> <tr> <td>CD163 Monoclonal Antibody (TNKUPJ), eBioscience™</td> <td><a href="https://www.thermofisher.com/antibody/product/CD163-Antibody-clone-TNKUPJ-Monoclonal/14-1631-82?icid=BID_FCREG_PJT3273_14-1631-82">14-1631-82</a></td> </tr> <tr> <td>CD163 Monoclonal Antibody (TNKUPJ), PerCP-eFluor 710, eBioscience™</td> <td><a href="https://www.thermofisher.com/antibody/product/CD163-Antibody-clone-TNKUPJ-Monoclonal/46-1631-82?icid=BID_FCREG_PJT3273_46-1631-82">46-1631-82</a></td> </tr> <tr> <td>CD163 Monoclonal Antibody (TNKUPJ), PE, eBioscience™</td> <td><a href="https://www.thermofisher.com/antibody/product/CD163-Antibody-clone-TNKUPJ-Monoclonal/12-1631-82?icid=BID_FCREG_PJT3273_12-1631-82">12-1631-82</a></td> </tr> </tbody> </table> <p><span style="font-size: medium">Search the entire catalog for all available <a href="https://www.thermofisher.com/antibody/primary/query/TNKUPJ?icid=BID_FCREG_PJT3273_CD163-clone-TNKUPJ">CD163 (clone TNKUPJ) monoclonal antibodies</a>.</span></p> <p>Interested in reading additional Behind the Bench blog posts for Flow Cytometry? See <a href="https://www.thermofisher.com/blog/behindthebench/tag/flow-cytometry/?icid=fl-flowblog">blog posts for flow cytometry</a>.</p> <p>For Research Use Only. 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