Immunophenotyping with DS8RELM, an Anti-Mouse RELM alpha Monoclonal Antibody for Studying Macrophages by Flow Cytometry

Are you studying macrophage polarization? Read this blog post to learn about the expression of the RELM alpha antigen on macrophages and the specific RELM alpha, clone DS8RELM antibody.

Dariusz Stepniak, Ph.D.
Dariusz, a Staff Scientist in R&D at Thermo Fisher Scientific,  was responsible for the development of this new antibody specificity. He spoke to us about the anti-mouse RELM alpha antibody (clone DS8RELM) for use with macrophages and shared insights into the performance and tips for use of antibody conjugates in multiplex, immunophenotyping flow cytometry applications.

Please tell me about the RELM alpha antigen.

RELM alpha also known as (Resistin-like alpha or FIZZ1) is a small cytokine (88 amino acids, MW 9.4 kDa) produced mostly by macrophages.¹ Its highest constitutive expression has been observed in small peritoneal macrophages, alveolar macrophages and adipose tissue macrophages. It is strongly induced by IL-4 and IL-13. Together with Arginase 1 and Ym-1 RELM alpha is considered one of the hallmarks of the M2 polarization of mouse macrophages, however, macrophages polarized with IL-10 (M2c) do not express neither RELM-alpha nor Arginase 1. Also, the expression patterns of RELM alpha and Arginase 1 in untreated tissue macrophages significantly differ. Elevated levels of RELM alpha may indicate parasitic infection. Humans do not have RELM alpha gene but human Resistin resembles mouse RELM alpha in respect to sequence, tissue distribution and function. Other members of RELM family in mouse: RELM beta, RELM gamma and Resistin display different tissue distribution and functional properties. RELM-alpha plays an important role in parasite infection, mucosal inflammation and allergy.

What type of research can utilize anti-mouse RELM alpha, clone DS8RELM?

Although our anti-mouse RELM alpha antibody might be of interest to all researchers studying macrophage biology, it will be particularly valuable to the scientists who aim to better understand the process of macrophage polarization. In addition, this new antibody will be useful for the researchers whose work focuses on parasitic infections.

If incorporating anti-mouse RELM alpha, clone DS8RELM into a flow cytometry panel, what other markers would be recommended for use and why?

Our monoclonal antibody DS8RELM recognizes mouse RELM alpha and can be added to any panel aiming to characterize macrophages. This DS8RELM antibody can be used with the below non-exhaustive list of mouse markers (Table 1).

Table 1. Non-exhaustive list of markers that can be used with DS8RELM.

Figure 1. Flow cytometric analysis of anti-mouse RELM alpha clone DS8RELM on wild type and RELM alpha knock-out (KO) macrophages. Thioglycollate induced macrophages from either C57BL/6 wild type mouse (purple histogram) or RELM-alpha knockout mouse (blue histogram) were stimulated with IL-4. As expected, clone DS8RELM stained a fraction of wild type cells but did not stain knock-out cells. Details: Thioglycollate induced macrophages from either C57BL/6 wild type mouse (purple histogram) or RELM-alpha knockout mouse (blue histogram) were stimulated in vitro with IL-4 for 48h. The cells were then surface stained with MHCII (clone M5/114.15.2) Alexa Fluor 700 and intracellularly stained with RELM alpha (clone DS8RELM) APC using the Intracellular Fixation & Permeabilization Buffer Set and protocol. MHCII positive cells were used for analysis. Data courtesy of Dr. Jiang Li, Meera Nair lab, University of California Riverside.

Figure 2. Immunophenotyping with the anti-mouse RELM alpha, DS8RELM antibody.

C57BL/6 mouse resident peritoneal exudate cells were surface stained with F4/80 Monoclonal Antibody, eFluor 450. The cells were then fixed and permeabilized using the Intracellular Fixation & Permeabilization Buffer Set and protocol, and intracellularly stained with either 0.25 µg of Rat IgG1 kappa Isotype Control, PE (left) or 0.25 µg of RELM alpha Monoclonal Antibody, PE (right). All cells were used for analysis.

What can you tell me about the relative expression of anti-mouse RELM alpha, clone DS8RELM in macrophages?

The highest level of constitutive expression of RELM alpha in mouse has been observed in Small Peritoneal Macrophages. In turn, Large Peritoneal Macrophages are known to express little or no RELM alpha unless they get stimulated. Il-4 and IL-13 are known factors that strongly induce RELM alpha expression. We have not observed any expression of this cytokine by lymphoid cells.

The expression of anti-mouse RELM alpha in mouse is medium to high compare to bright markers like CD4 or CD8.

Figure 3. Relative expression of RELM alpha using both PrimeFlow RNA assay and clone DS8RELM. Mouse resident peritoneal macrophages were stained to detect RELM alpha RNA (upper panels) or RELM alpha protein (lower panels). As expected based on known relative expression patterns clone DS8RELM stains the majority of Small Peritoneal Macrophages (both RNA (top right) and protein (bottom right)) and only a small fraction of Large Peritoneal Macrophages (same panels on the right). Details: C57BL/6 resident peritoneal cells were stained with CD11b (clone M1/70) APC and F4/80 (clone BM8) eFluor 450. The cells were then either submitted to the PrimeFlow RNA Assay and stained to detect RELM alpha mRNA (upper panels), or fixed and permeabilized using the Intracellular Fixation & Permeabilization Buffer Set, followed by staining with Rat IgG1 Isotype Control or RELM alpha (clone DS8RELM) PE  to detect RELM alpha protein (lower panels). In both cases CD11b positive cells were used for analysis.

Any tips and tricks you recommend for using anti-mouse RELM alpha, clone DS8RELM in flow cytometry panels?

Clone DS8RELM works best when used with Intracellular Fixation & Permeabilization Buffer set.

Is the anti-mouse RELM alpha, clone DS8RELM available for use in other applications?

This is being tested by our collaborators. We should soon know if the clone works in WB and IHC.

What key references should I review if I want to learn more about anti-mouse RELM alpha, clone DS8RELM?

Alternatively activated macrophage-derived RELM-{alpha} is a negative regulator of type 2 inflammation in the lung. J Exp Med. 2009; 206 (4): 937-52.

CD301b(+) Mononuclear Phagocytes Maintain Positive Energy Balance through Secretion of Resistin-like Molecule. Alpha. Immunity. 2016; 45 (3): 583-596.

High expression of RELM-α correlates with poor prognosis and promotes angiogenesis in gastric cancer. Oncol Rep. 2015; 34 (1): 77-86.

Resistin-like molecule-α regulates IL-13-induced chemokine production but not allergen-induced airway responses. Am J Respir Cell Mol Biol. 2012; 46 (5): 703-13.

Table 2. Anti-mouse RELM alph (DS8RELM ) monoclonal antibodies.

Search the entire catalog for all available RELM alpha (clone DS8RELM) monoclonal antibodies.

Interested in learning about other products or reading about other topics for flow cytometry? Read more Behind the Bench blog posts for Flow Cytometry here.

For Research Use Only. Not for Use in Diagnostic Procedures.

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