Investigating Immune Cell Killing

Exploring the role of the patient’s own immune system in defending the body against tumors is of paramount importance.  A critical component of this anti-cancer response is the ability of certain immune cells, such as cytotoxic T and natural killer cells, to induce malignant cell death through the process of immune cell killing (ICK). Modeling ICK in vitro is therefore of paramount importance. There are multiple techniques traditionally used to assess ICK, such as traditional flow cytometry and biochemical readouts, however, while valuable tools, they give limited insight into the dynamic interplay of cells.

With Incucyte® Immune Cell Killing Assays, visualizing, quantifying and understanding the dynamic interactions of immune and cancer cells has never been easier. Complement your existing immune or immuno-oncology protocols and start gaining new insight into immune cell, antibodies function & killing using non-invasive, non-disruptive analysis platform of cell health, phenotype, subsets and function.

Incucyte® Immune Cell Killing Assays

Incucyte Immune Cell Killing Assays allows for direct, multiplexed measurements of immune cell-mediated killing of tumor cells via the combination of real-time, automated analysis along with non-perturbing, live-cell reagents – all within your tissue culture incubator. A flexible assay platform ideal for:

• Cytotoxic T cell killing
• Antibody-dependent cell-mediated cytotoxicity (ADCC)
• Immune cell killing in tumor spheroid models

Incucyte® Immune Cell Killing Assay Concept

1. Target tumor cells are co-cultured with your immune cells of choice (e.g., T cells or human peripheral blood mononuclear cells; PBMCs).
2. Tumor cell death is measured directly and in real-time by adding the mix-and-read Incucyte® Caspase-3/7 Green or Caspase-3/7 Red Reagents or Incucyte® Annexin V Red or Green Reagents to your cultures.
3. Automated image analysis enables selective quantitation of tumor cell death. Optional: Simultaneous tumor cell counts can be enabled by labeling target cells with a range of Incucyte® Nuclight Labeling Reagents. Difficult to label cells can be identified using Incucyte® Cytolight Rapid Red or Incucyte® Cytolight Rapid Green Reagents.
4. Analysis with the Incucyte® Cell-By-Cell Analysis Software Module (Cat. No. 9600-0031) enables the quantification of both the labeled target cell population and the non-labeled effector cell population (proliferation and death). With adherent target cells, the software enables the user to track effector cell populations in the presence of target cells.

Key Advantages of the Incucyte® Immune Cell Killing Assays

Quantify real-time, dynamic cell interactions

Observe and quantify the dynamic interplay between immune cells and cancer cells to reveal cell-cell interactions and immunological synapse in complex co-culture models.

Figure 1A. 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.

Figure 1B. Visualize and quantify immune cell interactions with tumor cells within a mixed culture using Incucyte® Fabfluor-488. Cytolight Red A549 tumor cells were mixed with either pre-activated or non-activated PBMCs in the presence of Incucyte® Fabfluor-488-α-CD45 and Incucyte® Opti-Green to label the total lymphocyte population. Images at 2 hours post PBMC addition, show interactions between CD45+ cells (green) and A549 cells (red). Quantification of the interaction (overlay, yellow mask in images) reveals a marked increase in the interaction of activated effector cells with the target cells indicating cell engagement for immune killing of tumor cells (as shown in the bar graphs). This analysis was performed using the Incucyte® Cell-By-Cell Analysis Software Module.

Analyze tumor cell death and viability

Quantify tumor cell viability and death or immune cell proliferation using non-perturbing reagents and intuitive Incucyte® image analysis tools.

Figure 2A. Measure adherent tumor and effector cell proliferation, death and subpopulation analysis using Incucyte® Immune Cell Killing Assays. A549 cells, transduced with Incucyte® Nuclight NIR Lentivirus, were co-cultured with increasing numbers of activated (top row) or non-activated PBMCs (bottom row) in the presence of Incucyte® Annexin V Orange Reagent, Incucyte® Fabfluor-488-α-CD45 antibody complex and Incucyte® Opti-Green background suppressor.  Quantification of NIR (blue) nuclei indicates target cell proliferation and area of orange fluorescence (Annexin V), target cell death. Using Incucyte® Cell-by-Cell Analysis Software Module total effector cell numbers, in the presence of tumor cells can be quantified label free. The addition of Fabfluor-CD45 demonstrates that >80% are CD45 positive immune cells.

Figure 2B. Measure non-adherent target and effector cell proliferation, death and subpopulation analysis using Incucyte® Cell-by-Cell Analysis Module. Ramos cells, transduced with Incucyte® Nuclight Orange Lentivirus Reagent were co-cultured with activated or non-activated (using CD3/IL2) PBMCs in the presence of Incucyte® Annexin V NIR Reagent, Incucyte® Fabfluor-488-α-CD8 antibody complex and Incucyte® Opti-Green background suppressor. Quantification of Orange (red) nuclei indicates target cell proliferation and area of NIR (blue) fluorescence the target cell death. Using Incucyte® Cell-by-Cell Analysis Software Module total target and effector cell numbers, apoptotic index of target cells can be quantified and graphed over time.

Study immune-cell killing using a highly flexible assay format

Evaluate a wide range of relevant models including PBMCs, cytotoxic T lymphocytes and NK cells co-cultured with adherent or suspension tumor cell types. Use your choice of effector and target cells in T cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) formats and visualize and quantify Immune Cell Killing in Tumor Spheroid Models over time.

Figure 3A. ADCC: Trastuzumab (Herceptin®) induced immune cell killing of SKOV-3 ovarian cancer cells in immune-cell killing spheroid model: HER2-positive SKOV-3 Nuclight Red spheroids (2.5K/well) were seeded with PBMCs (6.25K/well) and treated with Herceptin (mAb targeting HER2 receptors), then imaged over 10 days (A). Herceptin induced inhibition of SKOV-3 spheroid growth was evaluated via fluorescence intensity (B).

Unlock your productivity with optimized protocols

Evaluate immune cell killing in both 96- and 384-well formats without the need for washing, cell lifting, or the use of radioactive labels.

Figure 4A. Simplify your workflow and maximize your insight. Combine the power of automated image and analysis with lab-tested protocols for both adherent or non-adherent target tumor cells in 2D or 3D co-culture models.

Commonly used immune cell killing assays

Common methods used to assess immune cell killing of cancer cells are often end-point, require cell lifting (e.g., flow cytometry) or measure indirect read outs of tumor cell viability (e.g., LDH, GAPDH release assays) or immune cell activation (ELISpot). All are generally non-image based. The table below shows the capabilities and challenges of some common approaches.

	Incucyte immune cell killing assays	Flow cytometry	51Cr release assay	GAPDH/LDH-release assay	DELFIA® TRF Assays	ELISpot	ADCC reporter bioassay (Promega)
Real-time cell visualization
Integrated analysis
Direct measurement of tumor cell death
Tumor cell viability measurements
Assess long term killing >24h
Flexible choice of target and effector cells
ADCC and T cell killing formats
Mix and read, no wash
384-well throughput
Cells can be used for further analysis
High sensitivity
Non-radioactive
No labeling antibodies

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