Macrophage Cell-based Assays
Immuno-Oncology Platform

Macrophage Cell-based Assays

Macrophage cell-based assays are employed to assess macrophage function, focusing especially on their contribution to the tumour microenvironment. In the context of cancer, tumour-associated macrophages often develop an anti-inflammatory phenotype that supports cancer progression, making them a novel target for emerging immunotherapies. These assays provide an valuable tool for in vitro analysis of macrophage function by:

  • Assessing macrophage polarization
  • Measuring macrophage phagocytosis
  • Evaluating target cell killing and inflammatory cytokines release by activated monocytes
  • Analysing the secretion of cytokines

Our suite of four macrophage cell-based assays offers a robust platform for evaluating the impact of your next generation immunotherapeutics on macrophage or monocyte function.

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Strategies of Immunotherapies
to Target Macrophages

Macrophages are highly plastic, with their polarization into the M1 subtype leading to an anti-tumour phenotype, and into the M2 subtype resulting in a pro-tumour phenotype. Approaches to target immunosuppressive macrophages include:

  • Inhibiting the effect of tumour promoters like IL-1b, complement, inflammasomes, myeloid checkpoints, and other inhibitory receptors that encourage immunosuppression.
  • Promoting anti-tumour effectors, by targeting receptors that stimulate the tumour-killing abilities of macrophages, such as CD40, STING, and Toll-like receptors

Reaction Biology’s macrophage-cell based assays are designed to test the efficacy of immunotherapeutics that employ these strategies on macrophages

Macrophage-cell Based Assays Available at Reaction Biology

  • Macrophage Polarization assay
  • Phagocytosis assay
  • Monocyte killing assay
  • Multiplex cytokine assay
Macrophage Polarization assay

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 assay

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 cytokine assay

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