Myeloid Cell In Vitro Assays

Myeloid Cell In Vitro Assays

 

Monocytes are a key component of the innate immune system with multiple functions. Typically, in response to inflammation, monocytes are rapidly recruited from the blood to the tissues where they will differentiate into either dendritic cells or macrophages. These phagocytic cells serve to present antigen to T cells, activating to mount a response against the offending antigen. In the tumour microenvironment, DCs and macrophages can be potentially targeted to increase or reignite the hyporesponsive immune response to the proliferating tumour. Aquila can design specific functional assays to understand the mechanics of antibody/compound interactions in the tumour milieu.

Dendritic Cells
Dendritic Cells (DCs) can be screened as primary cells or functionally in combination with T cells under mixed lymphocyte reaction (MLR) protocols. DC assay readouts can include determining the activation marker status by flow cytometry (CD80, CD83, CD86) as well as cytokine expression (IL-12, IL-10, TNF-α, IFN-γ, IL-6 etc.).

Characterisation of Macrophages
Macrophages are often found in tumours in large quantities. In the tumour microenvironment (TME), macrophage cells acquire pro-tumoral phenotype and are more commonly termed Tumour Associated Macrophages (TAMs). This phenotype is immunosuppressive (M2) rather than the normal inflammatory M1 phenotype. In the TME, TAMs can induce proliferation and survival of tumour cells, facilitate angiogenesis, and supress immune responses via expression of inhibitory molecules (eg. PD-L1, B7-H4) and cytokines (eg. IL-10, TGF-b). TAMs show mostly characteristics of an M2-like phenotype. This polarisation state is a consequence of expression of M2 signals by the tumour.

Aquila’s TAM “Re-Education” Assays:
Monocytes polarised with M-CSF, and post-stimulation with IL-4 or other factors such as IL-13, IL-10 and TGF-β, develop an M2 phenotype. These are defined by expression of CD68, 25F9, CD163 and CD209 markers, as well as their ability to produce IL-10, and low IL-12 in response to LPS. In contrast, M1 macrophages have the opposite cytokine balance where the IL-12 response dominates. Therapeutically, it is desirable to “re-direct” M2 macrophages towards an M1 (anti-tumour) phenotype. Aquila has a suite of M2 protocols which can be used to screen libraries of small molecules or antibodies. This allows interpretation of the therapeutic relevance of a shift of IL-12low/IL-10high responses of M2s to IL-12high/IL-10low.

Assay Read-Outs:

  • Monocyte to macrophage differentiation rate
  • Viability/apoptosis
  • Expression of M2 markers or inhibitory molecules by flow cytometry, qPCR, and cytokine release by ELISA or mulitplex

In addition to the standard M2 assays, Aquila is currently validating protocols using tumour conditioned media (cell lines and ovarian ascites) as well as isolating TAMs from ascites of ovarian cancer patients (Human Tumour Ex Vivo Systems).

Contact us to discuss your DC or macrophage assay study requirements.

CONTACT US

We’d be happy to discuss any service or product you may be interested in.

Aquila BioMedical Ltd.
Nine, Edinburgh BioQuarter
9, Little France Road
Edinburgh
EH16 4UX
UK

Tel: +44 (0) 131 658 5164
Email: info@aquila-bm.com

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