CD47/SIRPα Pathway Targeting
Evaluate “don’t eat me” signal blockers that enhance macrophage phagocytosis of tumor cells; quantify ADCP when combined with tumor-targeting antibodies
Macrophage Cell-Based Assays
Profile macrophage polarization, phagocytosis, and inflammatory responses
Evaluate how therapeutic candidates modulate macrophage phenotype and function—from M1/M2 polarization dynamics to tumor cell phagocytosis and cytokine secretion.
Targeting Macrophage Biology in Disease
Macrophages are highly plastic cells that adopt diverse functional states in response to environmental cues. Although the classical M1/M2 framework is a useful conceptual model, in vivo, macrophage phenotypes exist along a continuum of activation states.
M1-like macrophages, which are induced by IFN-γ or TLR ligands such as LPS, exhibit pro-inflammatory and tumoricidal activity. In contrast, M2-like macrophages, which are driven by IL-4, IL-13, or IL-10, display anti-inflammatory functions that can support tissue remodeling and tumor progression. In the tumor microenvironment, tumor-associated macrophages (TAMs) often exhibit immunosuppressive, M2-like characteristics that promote tumor growth, angiogenesis, and the suppression of anti-tumor immunity.
Therefore, targeting TAMs has emerged as a promising therapeutic strategy, including approaches that reprogram macrophages toward pro-inflammatory states or enhance their effector functions, such as CD40 agonists, STING pathway activation, and blockade of inhibitory signals like CD47.
How Macrophage Cell-Based Assays Work
- CD14+ monocytes isolated from PBMCs and differentiated into M0 macrophages using M-CSF over 6 days
- M1 polarization induced with IFN-γ and LPS; M2 polarization with IL-4 and IL-13 (or IL-4, IL-10, and TGF-b)
- Phenotype confirmed via surface markers: M1 (CD80+, CD86+, HLA-DR+) vs. M2 (CD163+, CD206+, CD209+)
- Functional readouts include phagocytosis and efferocytosis (flow cytometry, Cytation 5 real-time imaging), cytokine secretion (MSD multiplex), and T cell suppression in co-culture
- Test compounds evaluated for polarization shifts, enhanced phagocytosis and efferocytosis, or reversal of M2-mediated immunosuppression
Immunotherapy Strategies We Support
TAM Reprogramming Agents
Test STING agonists, TLR ligands, CD40 agonists, and CSF1R inhibitors for M2-to-M1 phenotype conversion
Checkpoint Inhibitor Evaluation
Measure how anti-PD-1/PD-L1 affects macrophage-mediated T cell suppression in co-culture systems
Beyond Oncology Applications
Assess immunomodulatory compounds for fibrosis, autoimmune, and inflammatory indications where macrophage dysfunction drives pathology
Flexible Assay Formats
M1/M2 Polarization Assays
- Drive monocyte-derived macrophages toward M1 or M2
- Characterize phenotype via surface markers (CD80, CD86, CD163, CD206, CD209, HLA-DR)
- Measure polarization-associated cytokines (IL-6, IL-10, IL-12, TNF-α, CCL22, IL-1b, IL-18)
- Assess compound effects on polarization state
Macrophage Phagocytosis Assays
- Quantify engulfment of labeled tumor cells or bioparticles
- Flow cytometry and real-time imaging (Cytation 5) readouts
- Evaluate anti-CD47 and other checkpoint blockers
- Compare phagocytic capacity across macrophage subtypes
M2 Suppression Co-Culture Assays
- Co-culture M2 macrophages with activated T cells
- Measure T cell suppression via IFN-γ release
- Test compounds for relief of M2-mediated immunosuppression
- Relevant for checkpoint inhibitor and TAM-targeting studies
Cytokine Secretion Profiling
- MSD multiplex quantification of inflammatory cytokines
- Profile M1 (IL-6, IL-12, TNF-α) vs. M2 (IL-10, CCL22) signatures
- Assess cytokine changes upon therapeutic treatment
- Sub-fg/mL sensitivity with minimal sample volumes
ADCP (Antibody-Dependent Cellular Phagocytosis)
- Quantify macrophage engulfment of antibody-opsonized targets
- Relevant for therapeutic antibody Fc optimization
- Compare to ADCC for comprehensive effector profiling
- Flow cytometry and imaging readouts available
Macrophage Efferocytosis Assays
- Quantify engulfment of labeled apoptotic normal and tumor cells
- Flow cytometry and real-time imaging (Cytation 5) readouts
- Evaluate anti-CD47 and other checkpoint blockers
- Compare phagocytic capacity across macrophage subtypes
Advantages of Our Platform
Phenotype and Functional Integration
Combine surface marker profiling with functional readouts (phagocytosis, cytokine secretion, T cell suppression) for comprehensive mechanistic characterization.
Primary Human Cells
Monocyte-derived macrophages from healthy donor PBMCs provide translational relevance that immortalized cell lines cannot match.
Multiple Readout Platforms
Flow cytometry for phenotyping, Cytation 5 for real-time imaging, MSD for cytokine multiplex—choose the readout that best fits your program.
Polarization Expertise
Validated protocols for generating M0, M1, M2, TAM subtypes enable precise testing of polarization-modulating compounds.
Beyond Oncology
Macrophage assays support therapeutic development in fibrosis, autoimmune disease, metabolic dysfunction, and infectious disease.
Applications and Case Studies
Macrophage Polarization
Test the effect of your immunotherapeutic compound on macrophage differentiation, within the tumour microenvironment or in response to inflammation and autoimmunity
Experimental workflow
- Isolation of CD14+ monocytes from healthy donor PBMCs
- Culturing cells under specific conditions to promote polarization into:
- Pro-inflammatory M1 macrophages
- Anti-inflammatory M2 macrophages
- Verification of the differentiation process through flow cytometry, identifying key surface markers such as CD86, CD163, and CD206 to distinguish macrophage phenotypes
- Combine cell phenotype with functional assays and cytokine measurements indicative of M1 and M2 types
Sample data
Phagocytosis Assay
Assess the phagocytic capabilities of various monocyte/macrophage subtypes utilizing labeled bioparticles
Experimental workflow
- Use of fluorescently tagged bioparticles, such as Zymosan-pHrodo and Ecoli-pHRodo
- Assessment of phagocytic activity by measuring internalization of flow cytometry analysis or real-time measurements using microscopy
- Comparing phagocytic capacity of various macrophage types or macrophage treatments
Sample data
Monocyte Killing
Investigate the efficacy of biologics by measuring their ability to kill target cells and induce monocytes to release inflammatory cytokines
Experimental workflow
- Isolation of CD14+ monocytes from PBMCs
- Co-culturing with target cells that express luciferase, at 10:1 (E:T ratio) effector to target ratio, meaning ten monocytes for every target cell
- Assessment of luciferase expression as a readout of viability of target cells
- Optional: Detection of inflammatory cytokine secretion in cell culture supernatants
Sample data
Multiplex Cytokines
Measure cytokines released upon differentiation or activation of immune response
Experimental workflow
- Isolation of CD14+ monocytes from PBMCs
- Co-culturing with target cells that express luciferase, at 10:1 (E:T ratio) effector to target ratio, meaning ten monocytes for every target cell
- Supernatant collection upon activation of immune response
- Cytokine level detection using electrochemiluminescent multiplex immunoassay (MSD)
Sample data
Frequently asked questions
How do you generate M1 and M2 macrophages?
CD14+ monocytes are isolated from PBMCs and differentiated into M0 macrophages using M-CSF over 6-7 days. M1 polarization is induced with IFN-γ and LPS; M2 polarization uses IL-4 and IL-13 or IL-4, IL-10, or TGF-b depending on the desired subtype. Phenotype is confirmed by surface marker expression and cytokine profiling.
What is the difference between ADCC and ADCP?
ADCC (antibody-dependent cellular cytotoxicity) involves NK cells killing antibody-coated targets. ADCP (antibody-dependent cellular phagocytosis) involves macrophages engulfing antibody-coated targets. Both are relevant for therapeutic antibodies; we offer assays for each mechanism.
Can you assess effects on tumor-associated macrophages specifically?
We can generate TAM-like phenotypes using tumor-conditioned media or specific cytokine cocktails. Effects on TAM reprogramming can be measured through phenotype shifts and functional changes.
What readouts demonstrate M2-to-M1 reprogramming?
Surface marker shifts (decreased CD163/CD206, increased CD80/CD86), cytokine profile changes (decreased IL-10, increased IL-6/TNF-α/IL-12), and restored T cell activation in co-culture assays all indicate successful M1 reprogramming.
Are macrophage assays relevant outside of oncology?
Absolutely. Macrophage polarization drives fibrosis (M2-like macrophages promote scarring), inflammatory diseases (M1-like drive tissue damage), and metabolic dysfunction. Our assays support therapeutic development across these indications.
Can you measure phagocytosis of specific tumor targets?
Yes, we use fluorescently labeled tumor cell lines or standard bioparticles as targets. For tumor cell phagocytosis, we can combine with anti-CD47 or tumor-targeting antibodies to assess ADCP activity.
Explore Macrophage-Targeting Strategies
From CD47 blockers to TAM reprogramming agents, our scientists will design a macrophage assay strategy aligned with your mechanism of action.