Immune cell clustering and proliferation assay

With the important role of the immune system on tumor progression, it’s thus crucial to characterize the possible impact of a drug candidate on the immune system. Through our Immune Cell Clustering and Proliferation assay we offers to assess phenotypically, in real time, the capacity of candidate compounds to promote immune cells activation when alone or in combination with T cell receptor (TCR) engagement. By catching the kinetics of the activation process, the assay allows to detect time-dependent effects that are overlooked in endpoint measurements.

Principle of immune cell clustering and proliferation live assay

Assay Principle

 

  • Immune cells of various origins (PBMCs, purified populations, etc) are exposed to an anti-CD3 antibody and potential immunomodulatory compound. Immune cell activation is then evaluated by phenotypical analysis.
  • Immune cell activation is monitored in real time by repeated scanning for every 2–4 hrs.
  • Immune cell proliferation is quantified using the confluence algorithm of the IncuCyte™ software.
  • Immune cell clustering is quantified by applying size filters to the confluence algorithm to identify aggregates – a key element when considering immune cell activation.

Representative Results

Immune cell proliferation

Figure 1. Representative images of PBMC proliferation. Phase-contrast images are overlaid with an IncuCyte ZOOM® confluence segmentation mask (yellow) of PBMCs activated with anti-CD3 antibody and IL-2. Images were taken at t=0 (A), 72 hours (B), and 144 hours (C). Cells were seeded at 15,000 cells/well. (D) Time course of PBMC proliferation in the presence of anti-CD3 antibody and IL-2, anti-CD3 antibody alone, and in the absence of activators.

Figure 1. Representative images of PBMC proliferation. Phase-contrast images are overlaid with an IncuCyte® confluence segmentation mask (yellow) of PBMCs activated with anti-CD3 antibody and IL-2. Images were taken at t=0 (A), 72 hours (B), and 144 hours (C). Cells were seeded at 15,000 cells/well. (D) Time course of PBMC proliferation in the presence of anti-CD3 antibody and IL-2, anti-CD3 antibody alone, and in the absence of activators.

Immune cell clustering

Figure 2. Activation of PBMCs with anti-CD3/IL-2 induces T-cell aggregation. Representative phase-contrast images of PBMCs activated with anti-CD3 antibody and IL-2 were taken at t=0 (A), 72 hours (B), and 144 hours (C) and overlaid with the IncuCyte ZOOM® confluence segmentation mask (blue), indicating cell clusters with an area greater than 1500 μm2 and a maximal eccentricity (non-circularity) of 0.95. (D) Representative time courses of T-cell subpopulation clustering when activated with IL-2 (10 ng/mL) and decreasing concentrations of anti-CD3 antibody.

Figure 2. Activation of PBMCs with anti-CD3/IL-2 induces T-cell aggregation. Representative phase-contrast images of PBMCs activated with anti-CD3 antibody and IL-2 were taken at t=0 (A), 72 hours (B), and 144 hours (C) and overlaid with the IncuCyte® confluence segmentation mask (blue), indicating cell clusters with an area greater than 1500 μm2 and a maximal eccentricity (non-circularity) of 0.95. (D) Representative time courses of T-cell subpopulation clustering when activated with IL-2 (10 ng/mL) and decreasing concentrations of anti-CD3 antibody.

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Explicyte's preclinical services in the field of high-content screening include kinetic cell-based-assays for the assessment of immune cell proliferation and clustering, using live-cell imaging, allowing to measure the ability of drug candidates to activate the anti-cancer immune response.