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.
- Time consuming 'end-point' fix and stain protocols that yield a single image per time-point
- Requires multiple washing steps and have to use radioactivity or antibody labelling for quantification
- Requirement to concatenate data from different samples (assay wells) to build time-courses, potentially introducing artefacts
- Loss of environmental control, resulting in cellular changes that may lead to unwanted and misleading experimental results
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.
Application
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
Time-lapse movies acquired using Incucyte® Live-Cell Analysis System showing real-time visualization of tumor cell killing in traditional 2D and more complex 3D models.
First, a T cell sub population of PBMCs, activated with anti-CD3 antibody and IL-2, is seen killing target SK-OV-3 tumor cells. Labeling of the SK-OV-3 cancer cells with the Incucyte® Nuclight Red Lentivirus enables direct counting of viable tumor cell numbers over-time. Addition of the Incucyte® Caspase-3/7 Reagent enables simultaneous detection of cells undergoing apoptosis (green fluorescence).
Next, Incucyte® Cell-by-Cell Analysis was used to quantify and graph total target and effector cell proliferation and the apoptotic index of target. Nuclight Red Ramos cells were cultured with activated human PBMCs in the presence of Incucyte® Annexin V Green Reagent, quantifying the subpopulation target cell proliferation, target cell death, and immune cell proliferation. Finally, the immune cell killing of A549 cancer cells in an immune-cell killing spheroid mode was observed and quantified. Nuclight Red A549 spheroids were cultured with non-activated and activated PBMCs.
- Target tumor cells are co-cultured with your immune cells of choice (e.g., T cells or human peripheral blood mononuclear cells; PBMCs).
- 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.
- 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.
- 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
- Quantify real-time dynamic interactions - Measure and observe the interplay between immune cells and cancer cells in complex co-cultures
- Analyze tumor cell death and viability - Selectively quantify tumor or immune cell proliferation and apoptosis using non-perturbing reagents and intuitive image analysis tools
- Study immune-cell killing using a flexible assay format - Evaluate a wide range of relevant immune and tumor cell co-culture models - in 2D or 3D
- Unlock your productivity with optimized protocols - Evaluate immune cell killing in 96- or 384-well plates using optimized reagents and protocols - no washing, no cell lifting, no radioactive labels
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).
Figure 3B. ADCC: Trastuzumab (Herceptin) induced immune cell killing of SKOV-3 ovarian cancer cells. PBMCs were co-cultured with either SKOV-3 Nuclight Red (HER2-positive) or A549 NucLight Red (HER2-negative) tumor cells. Time courses of trastuzumab-induced concentration-dependent inhibition of tumor cell proliferation in SKOV-3 (A) but not A549 (B) cells. Concentration-response curve to trastuzumab for inhibition of proliferation in SKOV-3 cells (C). Examples of unprocessed (top) and masked images (bottom) in the presence and absence of antibody (72h post addition, D).
Unlock your productivity with optimized protocols
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 | 51 Cr release assay | GAPDH/LDH-release assay | DELFIA® TRF Assays | ELISpot | ADCC reporter bioassay (Promega) | |
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| Real-time cell visualization |
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| Integrated analysis |
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| Direct measurement of tumor cell death | |||||||
Tumor cell viability measurements |
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Assess long term killing >24h | |||||||
Flexible choice of target and effector cells |
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ADCC and T cell killing formats |
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Mix and read, no wash |
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384-well throughput |
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Cells can be used for further analysis | |||||||
High sensitivity |
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Non-radioactive |
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No labeling antibodies |
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Griffiths, G.M. et al. Loss of ARPC1B impairs cytotoxic T lymphocyte maintenance and cytolytic activity. J Clin Invest, 129(12):5600-5614, 2019
- CD8 cytotoxic T lymphocytes (CTLs) rely on rapid reorganization of the branched F-actin network to drive the polarized secretion of lytic granules, initiating target cell death during the adaptive immune response. The Incucyte® Live-cell Analysis System was using to monitor loss of target cells over a longer time scale after IL-2 deprivation.
Adams, KJ et al. The use of induced pluripotent stem (iPS) cells for the safety testing of enhanced affinity TCR-transduced T cells. Cancer Research, AACR; April 5-9, San Diego, CA, 2014
- Testing off-target T cell activity against a panel of iPS cultures representing major organs of the human body. The Incucyte® system enabled direct visualization of caspase-3/7 dependent apoptosis, in real-time over a four day time course.
“Redirected killing by ImmTACs can be missed in short-term killing assays such as 51Cr-release and even the extended LDH-release assay. The adaptation and use of the Incucyte™ technology is therefore of critical importance for assessing ImmTAC potency and specificity under the strictest possible conditions”.
Adams, K. Redirected T cell activity by high affinity TCR-Anti-CD3 Bispecific candidate therapeutics. PhD Dissertation, Cardiff University (2013)
Ordering Information
Product | Qty. | Cat. No. |
|---|---|---|
1 module | ||
| Incucyte® Caspase-3/7 Green Reagent | 20 µl | |
| Incucyte® Caspase-3/7 Red Reagent | 20 µl | |
1 vial | ||
1 vial | ||
1 vial | ||
1 vial | ||
0.2 mL | ||
0.6 mL | ||
0.2 mL | ||
0.6 mL | ||
0.2 mL | ||
0.6 mL | ||
0.2 mL | ||
0.6 mL | ||
0.2 mL | ||
0.2 mL | ||
1 vial (15 µg) | ||
5 vials x 50 µg | ||
1 vial (1 mg) | ||
Incucyte® Mouse IgG2a Fabfluor-488 Antibody Labeling Reagent | 1 vial (50 µg) | |
Incucyte® Mouse IgG2b Fabfluor-488 Antibody Labeling Reagent | 1 vial (50 µg) | |
1 vial (50 µg) |
