What Is Immune Cell Killing?

Immune-cell recognition and killing of unwanted target cells, such as emergent tumor cells, is a critical component of the human host defense mechanism. Antibody-dependent cell-mediated cytotoxicity (ADCC) and T cell killing are two mechanisms of cell-mediated immune response. Each of these processes involves the stimulation of immune cell sub-populations, such as natural killer (NK) cells or cytotoxic T lymphocytes (CTL), which then actively lyse target cells. Understanding the interplay between immune and cancer cells and restoring and promoting the immune system’s capacity to fight and eliminate tumors ("cancer immunotherapy" or "immuno-oncology") is an exciting and promising research field.

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Introducing the IncuCyte® Immune Cell Killing Assays

An integrated solution for real-time visualization and automated analysis of immune cell-mediated killing of tumor cells – all within your tissue culture incubator. 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 IncuCyte® 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 Live-Cell Labeling Reagents. Difficult to label cells can be identified using IncuCyte CytoLight Rapid Red or IncuCyte CytoLight Rapid Green Labeling Reagents.
4. Analysis with the Cell-By-Cell Analysis Software Module (Cat. No. 9600-0031)
5. 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

• Real-time visualization and fully automated analysis of tumor cell killing and immune cell-tumor cell interaction
• Direct measurements of tumor cell death and viability
• Make relevant measurements in a highly flexible assay format
• Mix-and-read 96/384-well assays suitable for screening

Real-time visualization and fully automated analysis of interaction and killing in immune cell-tumor cell models

• Observe and quantify the dynamic interplay between immune cells and cancer cells
• Reveal cell-cell interactions, enabling insight into immunological synapse between subsets of cells
• Measure tumor cell death and the inhibition of cell proliferation using non-perturbing reagents

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.

Visualize and quantify immune cell interactions with tumor cells within a mixed culture using IncuCyte FabFluor-488. A549 CytoLight Red 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 Cell-By-Cell Analysis Software Module.

Direct measurements of tumor cell death and viability

• Selectively quantify tumor cell proliferation and apoptosis using intuitive IncuCyte® image analysis tools
• Measure tumor cell apoptosis using the IncuCyte® Caspase 3/7 Apoptosis Reagent and tumor cell proliferation using IncuCyte® NucLight Lentivirus Reagents
• Monitor the full time-course of tumor cell killing within your cell culture incubator. Confidently perform long-term studies (>5 days)

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 (IncuCyte® NucLight Red labeled nuclei, outlined in blue). Kinetic readouts reveal the time dependence of treatment effects.

Measure non-adherent tumor cell death and proliferation and immune cell proliferation using Cell-By-Cell Analysis in real time. Ramos NucLight Red cells were co-cultured with non-activated and pre-activated (CD3/IL-2) PBMCs in the presence of Annexin V Green Apoptosis Reagent. Using Non-Adherent Cell-By-Cell Analysis Software, images were acquired at 20X every 2 hours. Populations classified with red fluorescence to identify target and effector cell populations. Target cell proliferation, target cell apoptotic index, and effector cell proliferation were calculated and graphed over time for increasing target to effector cell ratios.

Make relevant measurements in a highly flexible assay format

• IncuCyte immune cell killing protocols developed for 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
• Visualize and quantify immune cell killing in tumor spheroid models

Mix-and-read 96/384-well assays suitable for screening

• No washing, no cell lifting, no radioactivity, no antibody labeling
• Setup and walk away – fully automated image capture and analysis
• Compatible with flow cytometry and end-point biochemical measurements – simply harvest cells/supernatants when the assay is complete

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

Ordering Information

Product name	Product Data Sheet	Safety Data Sheet	Qty	Cat. No.
IncuCyte® Caspase-3/7 Green Apoptosis Assay Reagent			1	4440
IncuCyte® Caspase-3/7 Red Apoptosis Assay Reagent			1	4704
IncuCyte® NucLight Red Lentivirus (EF-1α, Puro)			1	4476
IncuCyte® NucLight Green Lentivirus (EF-1α, Puro)			1	4475
IncuCyte® NucLight Red Lentivirus (EF-1α, Bleo)			1	4478
IncuCyte® NucLight Green Lentivirus (EF-1α, Bleo)			1	4477
IncuCyte® CytoLight Rapid Green Reagent – for cell labeling			1	4705
IncuCyte® CytoLight Rapid Red Reagent – for cell labeling			1	4706
IncuCyte® Mouse IgG1 FabFluor-488 Antibody Labeling Reagent			1 vial (50 µg)	4745
IncuCyte® Mouse IgG2a FabFluor-488 Antibody Labeling Reagent			1 vial (50 µg)	4743
IncuCyte® Mouse IgG2b FabFluor-488 Antibody Labeling Reagent			1 vial (50 µg)	4744

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