Immunology T cells


Quantifying dynamic T-cell function with IncuCyte and iQue analysis

T-cells are critical in adaptive immunity, with unique surface receptors that allow T cells to sense and respond to diverse types of pathogenic organisms as well as for defense against unwanted target cells, such as emergent tumor cells. T-cell activation and proliferation is fundamental to regulating the effect and extent of the immune response, requiring dynamic models in order to understand T-cell biology in fighting infection or disease.

The IncuCyte® Live-Cell Analysis System and IntelliCyt iQue Advanced Flow Cytometry Platform can be used to evaluate and quantitate key functions of T cells, including activation and clustering, antibody internalization assays, chemotaxis, cytokine production, as well as the evaluation of complex co-culture models, such immune cell killing and transendothelial migration assays.

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Activation & clustering

Label-free detection of T cell aggregation. Activation of PBMCs induces T cell aggregation or "clustering". User-friendly IncuCyte® software enables accurate quantification of cell clustering over time (blue mask).  Varying the activation conditions yields differing levels of proliferation (% confluence measurement) and clustering (cluster count based on object size).

T cell activation assessment with the iQue Advanced Flow Cytometry Platform and ForeCyt software. Dose and temporal responses in the percentage of activated T Cells and the levels of secreted IFNγ among different treatments. Unique patterns of T cell activation markers observed for each compound tested.

Immune cell killing

Visualize immune cell/tumor cell interplay using IncuCyte immune cell killing assays. (1) Physical contact between a small cytotoxic T cell and a larger labeled tumor cell (red). T lymphocyte division. (2) Tumor cells under attack from a cytotoxic T lymphocyte: The "kiss of death". (3) Tumor cell cytoplasmic granulation immediately followed by caspase 3/7 labeling (green), nuclear condensation and cell death. (4) Tumor cell mitosis: One cell becomes two.

Immunology T Cells Fig 2b

Measure tumor cell death and proliferation (optional) in real time using IncuCyte immune cell killing assays. User-friendly IncuCyte® software enables direct image-based detection of apoptotic tumor cells (green objects, outlined in yellow) and real-time counting of live tumor cells (IncCyte® NucLight red labeled nuclei, outlined in blue). Kinetic readouts reveal the time dependence of treatment effects.

Temporal Target Cell and Cytokine Analysis. SKOV-3 NucLight Green cells were weeded with PMBCS and activated with CD3/CD28 Dynabeads. (a) Target cell count timecourse analyzed with IncuCyte. Temporal cytokine data for (B) IFNγ and (C) TNFα using only 10µl/ day of supernatant samples were quantified using Qbead component of TCA kit (Qbeads or Assay Builder: IFNγ, option 1 and TNFα option 2)/ (D) Concentration response curves for AUC from the time-courses of target cell count (IncuCyte) and cytokines (iQue3).

3D Immune cell killing

Immunology T Cells Fig 3

Measure ADCC in tumor spheroids. Blended phase and fluorescent images of SKOV-3 NucLight Red® spheroids in the presence immune cells, either untreated or in the presence of anti-CD3 (10 ng/ml) and IL-2 (10 ng/ml) or Herceptin (0.08 – 50 ug/mL). Cytotoxicity was quantified based on the red fluorescent intensity. Data demonstrates destruction of tumour spheroids by the activated T-cell population and a Herceptin-induced cell cytotoxicity.

Quantifying Chemotaxis; TEM

Immunology T Cells Fig 4a

Visualization of leukocyte extravasation. CD3/CD28 Dynabead activated primary T cells were seeded on a HUVEC monolayer cultured on fibronectin and images were acquired every minute using the IncuCyte® System. Yellow and orange arrows indicated leukocytes moving between HUVEC cells and eventually down the pores (blue circles) of the ClearView insert.

Immunology T Cells Fig 4b

Primary T cells extravasation toward CXCL12. CD3/CD28 Dynabead activated primary T-cells were seeded on a HUVEC monolayer cultured on fibronectin. The insert containing T cell:HUVEC monolayer co-cultures was then exposed to CXCL12 gradients at the indicated concentrations. Images were acquired every 30 minutes using the IncuCyte® ZOOM and phase analysis was performed. Analysis of pharmacological response was performed at t=6 hr. Each data point represents mean ± SEM, n=4.

Quantifying Ab internalisation

Specific internalization responses driven by cell surface antigen expression. Jurkat (T cell like) cells (30K/well) were treated with various IncuCyte® FabFluor labeled antibodies (4 µg/mL) to show the specific nature. HD phase and red fluorescence images were captured every 30 minutes using a 20x objective over 12 hours. Time course data demonstrates a response profile mattching the expected expression of the tested markers, and confirms that an internalization response is only present when the specific antigen is expressed; notably no internalisation of the anti-CD20 antibody, a B cell marker, was observed. All data shown as a mean of at least 4 wells ± SEM, time course data shown as normalized red area (red area/phase area).


  1. Clustering; Zumwalde et al, 2013 J Immunol. October 1; 191(7): 3681–3693. doi:10.4049/jimmunol.1201954.
  2. Immune cell killing; McCormack et al, 2013 Cancer Immunol Immunother. Apr; 62(4): 773–785. doi:10.1007/s00262-012-1384-4
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