Immune Cell Killing Assays for Live-Cell Analysis
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 세포사 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 유세포분석 and biochemical readouts, however, while valuable tools, they give 제한 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.
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Gain additional insights into the mechanism of immune cell killing by combining live cell analysis and 유세포분석 into a single workflow
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:
®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® 카스파제-3/7 Reagent enables simultaneous detection of cells undergoing 세포자멸사 (green fluorescence).
Next, Incucyte® Cell-by-Cell Analysis was used to quantify and graph total target and 작동세포 proliferation and the apoptotic index of target. Nuclight Red Ramos cells were cultured with activated human PBMCs in the presence of Incucyte® 아넥신 V Green Reagent, quantifying the subpopulation target cell proliferation, target 세포사, 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.
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.
(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 카스파제 3/7 labeling (green), nuclear condensation and 세포사. (4) Tumor cell mitosis: One cell becomes two.
® 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.
Quantify tumor cell viability and death or immune cell proliferation using non-perturbing reagents and intuitive Incucyte® image analysis tools.
®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® 아넥신 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 (아넥신 V), target 세포사. Using Incucyte® Cell-by-Cell Analysis Software Module total 작동세포 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.
® 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® 아넥신 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 세포사. Using Incucyte® Cell-by-Cell Analysis Software Module total target and 작동세포 numbers, apoptotic index of target cells can be quantified and graphed over time.
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 세포독성 and antibody-dependent cell-mediated 세포독성 (ADCC) formats and visualize and quantify Immune Cell Killing in Tumor Spheroid Models over time.
®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).
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).
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
Common methods used to assess immune cell killing of cancer cells are often end-point, require cell lifting (e.g., 유세포분석) 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
유세포분석
51 Cr release assay
GAPDH/LDH-release assay
DELFIA® TRF Assays
ELISpot
ADCC reporter bioassay (Promega)
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
Griffiths, G.M. et al. . J Clin Invest, 129(12):5600-5614, 2019
Adams, KJ et al. . , AACR; April 5-9, San Diego, CA, 2014
“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. (2013)
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Yes, as long as the 형광단 excitation and emission spectra are compatible with the Incucyte® fluorescence imaging channels (See technical specifications for your instrument configuration). Note that data quality may be compromised if 형광단 expression is unstable over time or results in heterogeneous labeling. We recommend our Incucyte® Nuclight Lentivirus Labeling Reagents, as they are ideally suited for long-term live-cell studies. Target cells, especially those that are difficult to transduce, can also be labeled with Incucyte® Cytolight Rapid Green, Red, or Orange Dyes.
There is no need to pre-label your target cells when using adherent tumor cells. Tumor 세포사 is detected using either the Incucyte® 카스파제-3/7 염료 or Incucyte® 아넥신 V 염료, which is simply added to your assay along with your treatments. Although there is no need to label your target cells, you will find that using our Incucyte® Nuclight Lentivirus or Incucyte® Cytolight Reagents to label your tumor cells will enable you to make simultaneous measurements of viable target cell counts in real time. If you are using suspension target cells, they will need to be pre-labeled. Measurements of non-adherent tumor cell killing are made by quantifying the reduction in target-cell fluorescence over time. For both adherent and suspension cells, labelling of both target and effector cells is necessary to perform Cell-By-Cell Analysis.
Yes. When measuring non-adherent target cell killing, the Cell-by-Cell Analysis Software Module enables the user to quantify both features of the labelled target cell population and the non-labelled 작동세포 population (proliferation and death). With adherent target cells, the software enables the user to track 작동세포 populations in the presence of target cells.
Yes. Simply harvest cells/supernatants when the assay is complete, and perform your choice of further analysis. Workflow protocols exist for use of this assay in combination with iQue T-cell activation kit (TCA) to capture 사이토카인 changes and activation states of effector cells.
Yes. We have successfully performed immune cell killing experiments in a 384-well format.
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