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.


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4th Edition! Live-Cell Analysis Handbook

A guide to real-time live-cell imaging & analysis

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Application Note:

Gain additional insights into the mechanism of immune cell killing by combining live cell analysis and flow cytometry into a single workflow

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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.

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

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

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

51 Cr 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

  

 

 

 


Cancer Immunotherapy Discovery - Incucyte® Publications

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 cellsCancer 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.

Incucyte® Cell-by-Cell Analysis Software Module

1 module

9600-0031

Incucyte® Caspase-3/7 Green Reagent

20 µl

4440

Incucyte® Caspase-3/7 Red Reagent

20 µl

4704

Incucyte® Annexin V Green Reagent

1 vial

4642

Incucyte® Annexin V Red Reagent

1 vial

4641

Incucyte® Annexin V Orange Reagent

1 vial

4759

Incucyte® Annexin V NIR Reagent

1 vial

4768

Incucyte® Nuclight Green Lentivirus (bleo)

0.2 mL

4626

Incucyte® Nuclight Green Lentivirus (bleo)

0.6 mL

4477

Incucyte® Nuclight Red Lentivirus (bleo)

0.2 mL

4627

Incucyte® Nuclight Red Lentivirus (bleo)

0.6 mL

4478

Incucyte® Nuclight Green Lentivirus (puro)

0.2 mL

4624

Incucyte® Nuclight Green Lentivirus (puro)

0.6 mL

4475

Incucyte® Nuclight Red Lentivirus (puro)

0.2 mL

4625

Incucyte® Nuclight Red Lentivirus (puro)

0.6 mL

4476

Incucyte® Nuclight Orange Lentivirus (puro)

0.2 mL

4771

Incucyte® Nuclight NIR Lentivirus (puro)

0.2 mL

4805

Incucyte® Cytolight Rapid Green Reagent

1 vial (15 µg)

4705

Incucyte® Cytolight Rapid Red Reagent

5 vials x 50 µg

4706

Incucyte® Cytolight Rapid Orange Dye

1 vial (1 mg)

4839

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

Incucyte® Mouse IgG1 FabFluor-488 Antibody Labeling Reagent

1 vial (50 µg)

4745


Resources

Immune Cell Killing FAQs

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Incucyte® Immune Cell Killing Assay Protocol

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Incucyte® Immune Cell Killing of Tumor Spheroids Assay Protocol

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ELRIG 2017: Incucyte® S3 3D Immune Cell Killing

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CIMT 2016: Quantitative live-cell imaging assays for immunotherapy

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White Paper: The Evolution of Immune Cell Killing Assays Using Live-Cell Analysis

Evolution of Immune Cell Killing (ICK) assays

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Live-cell Immunocytochemistry

Reveal the Dynamics of Surface Protein Expression with a New Approach to Immunocytochemistry

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Combined iQue - Incucyte workflow

In vitro cell biology models are becoming ever more complex to better mimic in vivo studies. However deciphering the meaning of results gained...

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