The number of IncuCyte® references has surpassed 2,000!
Sign up for the Live Cell Insights Publications eNewsletter to read new and notable research articles featuring the IncuCyte.
Thank you for signing up!
No thanks


IncuCyte® Immune Cell Killing Assays for Live-Cell Analysis

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.

Request more information    See all applications

3rd Edition! Live-Cell Analysis Handbook

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

Reserve your copy

Application Note:

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

Download Your Copy

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

T cell sub population of PBMCs, activated with anti-CD3 antibody and IL-2, killing target SK-OV-3 tumor cells

Time-lapse movies acquired using IncuCyte® Live-cell Analysis System showing a T cell sub population of PBMCs, activated with anti-CD3 antibody and IL-2, killing target SK-OV-3 tumor cells.

Labeling of the SK-OV-3 cancer cells with the IncuCyte® NucLight Red Live-Cell Labeling reagent enables direct counting of viable tumor cell numbers over-time. Addition of the IncuCyte® Caspase 3/7 apoptosis reagent enables simultaneous detection of cells undergoing apoptosis (green fluorescence).

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.

Cancer Immunotherapy Discovery - IncuCyte® Publications

McCormack E, Adams KJ, Hassan NJ, Kotian A, Lissin NM, Sami M, et al. Bi-specific TCR-anti CD3 redirected T-cell targeting of NY-ESO-1- and LAGE-1-positive tumors. Cancer Immunol Immunother, 62(4):773–85, 2013

  • Directing T cells to kill antigen expressing tumor cells using a novel T cell engaging protein. The IncuCyte™ system was used to selectively quantify the whole killing time course and revealed activities at timepoints that alternative methodologies could not detect.

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)

Key Advantages

Key Advantages of the IncuCyte® Immune Cell Killing Assays

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
  • Label-free quantification of effector cell proliferation or activation
Visualize immune cell/tumor cell interplay using IncuCyte immune cell killing assays

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.

Immune Cell Killing ICC Figure

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 in real time

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

3D Immune Cell Killing Video

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). Herceptin induced inhibition of SKOV-3 spheroid growth.

Watch the video

Download the protocol

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).

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

  • No washing, no cell lifting, no radioactivity, no antibody labeling
  • Setup andc 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 Assays

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
No labeling antibodies

Ordering Info

Tech Resources

No file selected
Stay up-to-date with what's happening at Essen BioScience

© 2020 Essen BioScience

Privacy Policy