As a leading service provider in the field of antibody discovery and immunotherapy development, Creative Biolabs now presents one-stop development services for CAR-T therapy, which has become a more promising approach among cancer therapies. We offer macrophage polarization assays used to improve the antitumor immunity via promoting inflammation during the one-stop service for further downstream research.

Macrophage Polarization

Macrophages belong to a heterogeneous population of innate myeloid cells involved in disease and health. They are the most functional diverse cells of the hematopoietic system. Macrophages exert multiple functions in humans, including responding to pathogens and modulating the adaptive immune response, induction and resolution of inflammation, tissue repair, and homeostasis. Macrophages own appealing plasticity characteristics. Therefore, in response to different stimuli, different populations of macrophages with various physiological and pathological roles can be developed. Creative Biolabs offers the macrophage polarization assay to induce different macrophage subsets.

Fig.1 Macrophage polarization.^1,2

Macrophage Polarization Assay Services at Creative Biolabs

Macrophages reveal specific phenotypes and functions in response to different triggers. In the macrophage polarization assay, we provide specific stimuli co-cultured with isolated bone marrow-derived or peritoneal macrophages to generate different polarized macrophage subsets. Depending on the types of stimuli that macrophages are exposed to, they are able to polarize to M1 (pro-inflammatory) or M2 (anti-inflammatory) macrophages.

M1 Macrophage Polarization Assay

Creative Biolabs can generate macrophages by treating isolated bone marrow-derived or peritoneal macrophages with specific stimuli. Our M1 polarization protocols are designed to achieve a stable, optimal, and effective regimen for in vitro induction to drive antigen-specific immune responses. Our M1 macrophage polarization assay allows the assessment of cytokine profiles, cell surface receptor expression, scavenging functions, and the ability to activate or suppress T-cell proliferation.

M2 Macrophage Polarization Assay

Based on our powerful platform, macrophages can be efficiently transformed into M2 and demonstrate effective phagocytosis. For the validation of M2 macrophages, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), flow cytometry (FC) are usually carried out to analyze surface markers and cytokines expression upon request. Our assay also offers a wide range of valuable candidates to assess the potential of drugs to disrupt the M2 polarization-induced signaling.

Based on the stimuli and the achieved transcriptional changes, the M2 macrophages can be classified into four subdivisions - M2a, M2b, M2c, and M2d. In this assay, except for different macrophage polarization by adding certain stimuli, we also provide support for our clients with verification of each polarized macrophage-based on the distinctly expressed surface markers and/or produced cytokines. Here we list the different stimuli used to trigger different macrophage populations.

Core Assay Technologies

• M1 macrophage: IFNγ and LPS
• M2a macrophage: IL-4 or IL-13
• M2b macrophage: Immune complexes and LPS
• M2c macrophage: Glucocorticoids or IL-10
• M2d macrophage: Adenosines or IL-6

FACS analysis

• Surface marker detection: identify M1 (CD80, CD86) and M2 (CD206, CD163) phenotypic features by fluorescent labeled antibodies.
• Intracellular factor detection: detection of TNF-α, IL-10 and other cytokine expression in combination with fixed permeabilization treatment.
• Data analysis: gating, compensation and statistical analysis using software such as FlowJo to output M1/M2 ratio and activation status.

Immunohistochemical staining

• Detection of M1 macrophages (e.g. LPS/IFN-γ induced samples) using iNOS antibody.

Functional tests

• Phagocytosis assessment: detect the phagocytic efficiency of macrophages on tumor cells by antibody-dependent cell phagocytosis (ADCP) assay.
• Immunosuppressive activity: to assess the inhibitory effect of tumor-associated macrophages (TAM) on T cell activation.

Related Products

On the basis of providing professional macrophage polarization assay services, we have developed a multi-dimensional supporting product system covering experimental materials, assay reagents and functional analysis tools to ensure the integrity and reliability of the research protocol.

We provide macrophages, monocytes, reagents for culturing, differentiation, and ELISA kits that analyze cytokines, chemokines, surface markers, and other proteins relevant to macrophage function and activation for our clients' research.

Service Features

Efficient Process Support

• Standardized testing solution: from cell isolation to polarization induction to functional testing, we provide complete experimental process support.
• Primary culture system: supporting M-CSF, LPS and other inducers and primary culture medium, reducing the cost of customers' purchases and improving the efficiency of experiments.

Product and Service Synergies

• Supporting reagents: specific antibodies are compatible with flow cytometry and immunohistochemistry assays, and functional validation tools are directly related to tumor immunology research needs.
• Scale-up testing capability: support large-scale sample testing, provide intelligent culture and testing equipment, reduce human error and improve data consistency.

Application Scenario Coverage

• Tumor immunity research: validate the polarization state and immunosuppressive function of TAM, and evaluate the correlation between M1 polarization persistence and anti-tumor efficacy in therapies.
• Chronic and autoimmune diseases: dynamic monitoring of macrophage polarization balance to assess disease progression and therapeutic efficacy.

Competitive Highlights

• Flexible customization capability: adjust induction conditions and assay specifications according to customer sample type (primary/cell line) and research objectives.
• Technology upgrade support: continuously follow up on new polarization-regulating materials and assays to ensure cutting-edge services.

Workflow of Macrophage Polarization Assay Services

Scientific Resources

Learn More Macrophage Biology

Learn More Macrophage Polarization

Learn More Different Approaches to Regulating Polarization of Tumor-Associated Macrophages

Learn More Isolation and Identification of Macrophages

Q & A

Q: Does the assay service cover a full range of markers for M1/M2?

A: Yes, we use a multi-dimensional detection system including: surface markers CD80/CD86 (M1), CD206/CD163 (M2), etc., and achieve high sensitivity identification, intracellular marker validation as well as functional validation by FACS.

Q: Are different sample types supported?

A: Yes, we support the detection of different samples, including:

• Primary cells: monocytes isolated from whole blood PBMCs are induced to differentiate to M0 and then polarized to M1/M2 by M-CSF.
• Cell lines: such as THP-1, RAW264.7, etc., which are directly induced to polarize.
• Tumor tissue sections or single cell suspensions: immunohistochemical analysis combined with flow-through assays to verify polarization status.

Q: Can you provide some considerations for cell line selection and culture?

A: Absolutely.

• RAW264.7 cell line: susceptible to LPS+IFN-γ induced M1 polarization and IL-4 induced M2 polarization, but need to be resuscitated if the polarization ratio is too high. Culture density is recommended to be 5×10⁶~1×10⁷/mL, and serum-free freezing solution is used to avoid batch differences.
• THP-1 cell line: can be induced to differentiate into macrophage after PMA treatment, and then LPS+IFN-γ to induce M1 type. IL-4/IL-13 induced to secrete TGF-β, IL-10, suitable for chronic inflammation and tissue repair studies.

Q: How long is the experiment cycle and what are the deliverables?

A: We have flexibility in our cycles. The exact timing depends on your cell line and the customization of the experiment. Deliverables usually include raw data, cell characterization results and a detailed lab report.

References

1. Zanluqui, N.G., et al. Macrophage polarization in chagas disease. J Clin Cell Immunol. 2015. http://dx.doi.org/10.4172/2155-9899.1000317
2. Under Open Access license CC BY 4.0, without modification.