Immune Cell Killing Assays

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Workflow

Liquid Tumors

Literature

Evaluate Immune Cell Killing in Real Time

xCELLigence RTCA uses label-free, impedance-based technology to continuously measure immune cell killing—from minutes to days—automatically. This enables real-time monitoring at low, physiologically relevant effector:target (E:T) ratios, providing highly sensitive, quantitative potency data.

Accurate, real-time measurements of immune cell killing potency.

Detailed insights into killing mechanisms, serial killing and exhaustion.

Simple workflows - seed cells and monitor killing kinetics under physiological conditions.

FDA 21 CFR Part 11 compliance for electronic data integrity.

Immune Cell Killing Assay Workflow

xCELLigence RTCA uses impedance-based technology to continuously and automatically measure immune cell killing from minutes to days without extra labelling. This high-throughput, user-friendly workflow enables precise monitoring at low, physiologically relevant effector:target (E:T) ratios, delivering sensitive, quantitative potency data.

Easy to use: Add target and effector cells to patented E-Plates, insert E-Plate into the xCELLigence instrument, and start monitoring:

Assay principle: adherent target cells grow on the gold biosensors and the cell index rises; non-adherent effector cells alone give no signal; after their addition the cell index falls, more steeply at higher effector-to-target ratios

Step 1: Adherent target cells (e.g. tumor cells) are seeded into E-Plate wells. Cell index increases as target cells attach and proliferate.

Step 2: Non-adherent effector cells (e.g. CAR T cells) are added to the wells. These cells do not lead to an increase in Cell Index since they do not adhere to the gold electrodes.

Step 3: Cytolytic activity of the effector cells is sensitively and precisely detected, represented by a decrease of the Cell Index.

Application Note: Real-Time, Label-Free Measurement of Natural Killer Cell Activity and Antibody-Dependent Cell-Mediated Cytotoxicity

Multiple Perspectives into Immune Cell Killing Activity

Researchers are continuously improving chimeric antigen receptor T cells to maximize specificity, potency, and persistence. The xCELLigence RTCA eSight combines impedance-based technology and live cell imaging to obtain highly sensitive, multiplexed and quantitative measurements of immune cell killing from multiple perspectives, without increasing the workload. Get more consistent results and save time by measuring immune cell killing in one workflow.

Multiplex Impedance-Based Data with Live Cell Imaging for Increased Confidence

While the standard xCELLigence RTCA systems (SP, MP, and HT models) offer comprehensive, non-invasive, label-free measurements in 96- or 384-well formats, the xCELLigence RTCA eSight combines this technology with live-cell imaging to further strengthen the reliability of your cell analysis and results. This integrated instrument enables you to capture five complementary data streams—impedance, brightfield, and red, green, and blue fluorescence imaging—on the very same cell population, providing deeper insights into immune-cell-mediated killing.

Application Note: Real-Time Potency Assay for CAR T Cell Killing of Adherent Cancer Cells

CAR T cell killing efficacy on the xCELLigence RTCA eSight: schematic of CD19-CAR T cells with HEK-293 control cells, HEK-293-CD19 cells and B cells; impedance trace in which the control grows to confluency while the CD19-positive target cells die after the CAR T cells are added; and brightfield images with red-labelled cells at an E:T ratio of 4:1 from 0 to 48 hours, in overview and enlarged

Immune Cell Killing Assays Targeting Liquid Tumors

The xCELLigence RTCA immune cell killing assay can be adapted to evaluate liquid cancer cells by pre-coating the E-plate wells with a tethering antibody from Immunotherapy Kits. Liquid tumor cells attach to the tethering antibodies, allowing cell health to be sensitively monitored by the impedance biosensors. Once immobilized cells reach confluency, effector cells are added and cell killing is measured across multiple effector-to-target (E:T) ratios, providing real-time, quantitative insights into immune-mediated cytotoxicity.

Application Note: Evaluating Functional Potency of Immunotherapies Targeting Tumors of B Cell Origin

Killing assay with suspension target cells

Deeper Insights Compared to Other Potency Assays

Unlike labor-intensive assays requiring multiple timepoints and techniques, xCELLigence RTCA offers a label-free, automated solution that delivers a comprehensive view of immune cell killing. It captures dynamic cell behavior at low, physiologically relevant E:T ratios and automatically generates detailed data plots using xCELLigence RTCA Software Pro, providing a more in vivo-like analysis.

Case study:
Building a CAR-T Toolbox for Cell Therapies

Comparison table of common potency assays: xCELLigence eSight and RTCA against live cell imaging, flow cytometry, chromium release, LDH release and IFN-gamma release, across label-free, handling steps, real-time kinetics, long-term killing, mechanism of action and sensitivity

Application Notes

Real-Time Potency Assay for CAR T Cell Killing of Adherent Cancer Cells

Bispecific T Cell Engager (BiTE) Mediated Cancer Cell Lysis

Evaluating Functional Potency of Immunotherapies Targeting Tumors of B Cell Origin

Real-time measurement of Natural Killer Cell Activity and Antibody-Dependent Cell-Mediated Cytotoxicity

Label-Free Assay for NK Cell-Mediated Cytolysis

Case Study

Case Study: Building a CAR-T Toolbox for More Comprehensive Assessment of Cell Therapies

Case Study: Immune-cell function - Quantifying Immune-Cell Mediated Killing

Handbook

xCELLigence RTCA Cancer Immunotherapy

Publications

  1. Nagarsheth, N.B.; Norberg, S. M.; Sinkoe, A. L. et al. TCR-Engineered T Cells Targeting E7 for Patients with Metastatic HPV-Associated Epithelial Cancers. Nat. Med. 2021, 27(3), 419–425. doi:10.1038/s41591-020-01225-1
  2. Rodriguez-Garcia, A.; Lynn, R. C.; Poussin, M. et al. CAR-T Cell-Mediated Depletion of Immunosuppressive Tumor-Associated Macrophages Promotes Endogenous Antitumor Immunity and Augments Adoptive Immunotherapy. Nat Commun. 2021, 12(1), 877. Published 9 Feb 2021. doi:10.1038/s41467-021-20893-2
  3. Nguyen, P.; Okeke, E.; Clay, M. et al. Route of 41BB/41BBL Costimulation Determines Effector Function of B7-H3-CAR.CD28ζ T Cells. Mol. Ther. Oncolytics. 2020, 18, 202–214. Published 23 Jun 2020. doi:10.1016/j.omto.2020.06.018
  4. Park, A. K.; Fong, Y.; Kim, S. I. et al. Effective Combination Immunotherapy Using Oncolytic Viruses to Deliver CAR Targets to Solid Tumors. Sci. Transl. Med. 2020, 12(559), eaaz1863. doi:10.1126/scitranslmed.aaz1863
  5. Gitto, S. B.; Kim, H.; Rafial, S. et al. An Autologous Humanized Patient-Derived-Xenograft Platform to Evaluate Immunotherapy in Ovarian Cancer. Gynecol. Oncol. 2020, 156(1), 222–232.
  6. Shrestha, B.; Zhang, Y.; Yu, B. et al. Generation of Antitumor T Cells for Adoptive Cell Therapy With Artificial Antigen Presenting Cells. Journal Immunother. 2020, 43(3), 79–88. doi:10.1097/CJI.0000000000000306
  7. Choi, B.D.; Yu, X.; Castano, A.P. et al. CAR-T cells Secreting BiTEs Circumvent Antigen Escape Without Detectable Toxicity. Nat. Biotechnol. 2019, 37, 1049–1058.

Protocols

  1. Dunsford, L. S.; Thoirs, R. H.; Rathbone, E.; Patakas, A. A Human In Vitro T Cell Exhaustion Model for Assessing Immuno-Oncology Therapies. In Immuno-Oncology. Methods in Pharmacology and Toxicology; Tan, S. L., Eds.; Humana, New York, NY, 2020.
  2. Xi, B.; Ye, P.; Golubovskaya, V.; Abassi, Y. In Vitro Functional Assay Using Real-Time Cell Analysis for Assessing Cancer Immunotherapeutic Agents. In Immuno-Oncology. Methods in Pharmacology and Toxicology. Tan, S. L. Eds.; Humana, New York, NY, 2020.
  3. Xi, B.; Berahovich, R.; Zhou, H. et al. A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells. J. Vis. Exp. 2019, 153, e59033, doi:10.3791/59033.

For Research Use Only. Not for use in diagnostic procedures.