Serial Killing Assay Using Longitudinal Impedance-Based Tumor Cell Viability Measurement – A Useful Method to Assess T Cell Performance

Chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer immunotherapy with remarkable clinical success across multiple hematologic malignancies such as chronic lymphoid leukemia, acute lymphoblastic leukemia, large B-cell lymphoma and multiple myeloma^1,2,3,4,5. Since the first regulatory approvals, the number of CAR T cell products and treated patients has expanded steadily⁶. Depending on the specific product and clinical indication, typical infusion doses range from approximately 2 × 10⁶to 5 × 10⁸CAR⁺ T cells per patient⁷. High infusion doses are necessary to achieve effective tumor clearance and long-term disease control, but are associated with clinical and practical challenges. Large cell numbers may increase the risk of serious side effects like cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), both of which can be dose-dependent⁸. In addition, manufacturing sufficient numbers of functional CAR T cells is challenging from heavily pretreated or lymphodepleted patients, and the process can be logistically demanding and costly for treatment centers⁹.

Despite these challenges, CD19-targeted CAR T cell therapies achieve 70%-90% complete remission rates in patients with relapsed or refractory B-cell malignancies, demonstrating a transformative clinical impact^10,11,12. However, CAR T cell therapy faces relevant challenges that limit its long-term efficacy. Key obstacles include antigen loss or downregulation, insufficient CAR T cell persistence and functional exhaustion⁹. CAR T cell exhaustion - characterized by decreased proliferative capacity, impaired anti-tumor activity, and poor persistence - represents a vital cause of nonresponse and relapse¹³. Exhausted CAR T cells come with reduced effector function, persistent expression of inhibitory receptors, and defective cytokine production due to chronic antigen exposure and immunosuppressive tumor microenvironments¹⁴. Clinical studies have demonstrated that loss of functional CAR T cell persistence is associated with an increased risk of relapse^11,15,16,17. Because prolonged in vivo activity appears to be a major predictor of therapeutic success, understanding the durability and sustained killing capacity of CAR T cell products is an important goal in both preclinical and translational research. Of particular interest is the ability of CAR T cells to repeatedly recognize and lyse tumor cells over extended periods - so called 'serial killing' - as this property provides a functional measure of persistence and resistance to exhaustion¹⁸.

This article provides a detailed protocol for performing serial killing assays using the xCELLigence Real-Time Cell Analysis (RTCA) impedance platform, including an optional washing procedure that enables re-use of assay plates, further referred to as E-plates, thus improving cost-efficiency without compromising assay performance. The rationale for developing such a protocol arises from limitations in conventional cytotoxicity assays, which typically offer only static, endpoint readouts. Common approaches, such as chromium-51 release, lactate dehydrogenase (LDH) assays, or flow cytometry-based viability assays require labels, involve labor-intensive workflows, and are limited to defined time points^18,19. More importantly, they cannot easily capture dynamic killing kinetics over prolonged stimulation or multiple rounds of antigen exposure, a situation directly linked to CAR T cell persistence and functional exhaustion¹⁸.

The xCELLigence RTCA platform addresses these gaps by providing label-free, real-time, kinetic monitoring of target cell viability via electrical impedance²⁰. Adherent tumor target cells are seeded on gold microelectrode-coated E-plates, where their attachment and proliferation increase the measured impedance, reported as the Cell Index (CI)²¹. Upon CAR T-mediated killing, target cells are killed and detach, leading to a rapid drop in impedance²⁰. This real-time readout allows continuous assessment of cytotoxicity over hours or days without the need for any additional labels or reagents²². CAR T cells, being non-adherent, do not contribute to the impedance signal, allowing for a clean separation of effector and target readouts²³. Because data are recorded continuously, researchers can conduct multi-round stimulation protocols where CAR T cells are repeatedly transferred and exposed to fresh tumor target cells, allowing direct measurement of how killing capacity evolves over time²⁴. This mimics the repeated antigen encounters CAR T cells experience in vivo and provides a functional readout of their cytotoxic capacities.

This impedance-based serial killing assay is ideally suited for researchers evaluating CAR T cell function in preclinical settings, including construct optimization, functional comparisons, and potency testing. This protocol requires the xCELLigence RTCA system and the use of E-Plate 96. While broadly applicable to adherent tumor models, it is not directly suited for suspension cultures without immobilization or tethering strategies. Because impedance measurements alone reflect only target-cell adherence and viability, complementary analyses such as flow cytometry or cytokine assays are recommended to fully characterize CAR T cell activation, proliferation, and exhaustion dynamics. The protocol includes designated steps that allow harvesting of cells and supernatants for these additional readouts, enabling a more comprehensive assessment of CAR T cell functionality.

NOTE: This protocol has been optimized for the E-Plate 96 and requires the xCELLigence RTCA system. Adherent tumor cells are seeded overnight in advance to reach the logarithmic growth phase at the time of CAR T cell addition. Depending on the tumor cell line and seeding density, this interval may be shortened to 6 - 9 h, allowing same-day effector addition. If the latter workflow is performed, the nominal day-by-day timeline described in the protocol is altered, and users should adapt the schedule accordingly. Co-cultures are maintained for 48 h before CAR T cells are transferred onto freshly plated tumor cells, requiring two plate positions on the xCELLigence instrument and alternating tumor cell plating and effector addition every other day. CAR T cells should be generated according to institutional SOPs or established protocols. Briefly, peripheral blood T cells are isolated under approved ethical standards, transduced under appropriate biosafety conditions, and assessed for transduction efficiency and viability by flow cytometry or equivalent QC assays. xCELLigence assays should not be performed when CAR transduction efficiency is below 20%, as lower efficiencies may lead to increased background and unspecific killing by untransduced cells, which can distort tumor growth curves. All cell handling should be performed under sterile conditions in a laminar flow hood. Media can be used to the laboratory's standard or as recommended by the supplier. Unless specified otherwise, cells are incubated at 37 °C in a humidified atmosphere containing 5% CO₂. While optimized for CAR T cells, this method can also be applied to other suspension-based effector cells and adherent targets.

1. Preparation and blanking (Day 1)

1. Plate preparation
   1. Add 50 µL of tumor medium to the bottom of each well of the E-plate 96.
      ​NOTE: Be careful not to scratch the bottom of the well as this might cause damage to the plate.
   2. Place the E-plate in the incubator, positioning it on the same machine slot to be used for measurements.
   3. Incubate the E-Plate for 30 min at 37 °C to bring it to the appropriate assay temperature and pH.
2. Instrument setup
   1. Meanwhile, launch the RTCA Software Pro (Version 2.6.1) and select a file path to save the experiment.
   2. Enter the experiment name in the "RTCA Operator" field and save.
   3. Select the wells to be measured.
   4. Assign cell names under the Cell tab and treatment types under the Treatment tab.
3. Measurement schedule
   1. Set up the schedule with the following steps:
      Step Name = Blank ; Sweeps = 1 ; Interval = 1 ; Duration = 0:00
      Step Name = Tumor Cells ; Sweeps = 250-300 ; Interval = 15 min; Duration = minimum 24 h
      Step Name = Treatment ; Sweeps = up to 2500 ; Interval = 5 min; Duration = minimum 72 h
   2. Press Apply and ensure that Auto is not selected for any step.
4. Blanking step
   1. After the 30 min incubation of the E-Plate at 37 °C, press the PLAY/RUN to initiate the blank step.
   2. Wait until the software marks the step DONE (approx. 1-2 min).
   3. Unlock the cradle and remove the E-plate only when ready to proceed with cell seeding.

2. Round 1: Tumor cell seeding (Day 1)

1. Seeding tumor cells
   1. Prepare a suspension of tumor cells in 50 µL of appropriate medium per well.
   2. Remove the E-plate from the incubator and seed the cells in triplicates.
2. Starting measurement
   1. Immediately return the E-plate to the machine and lock the cradle.
   2. Wait for 5 -10 min before the first measurement to equilibrate the E-plate temperature and pH, then click the PLAY/RUN button to start the measurement (Step 2 of schedule = Tumor Cells).
   3. Incubate the E-plate overnight to allow cell adherence. Monitor the non-normalized Cell Index (CI). When the CI enters the log-phase growth stage, proceed to the next step. This phase depends on the cell line used and can be influenced by the number of seeded cells. BxPC3 cells (ATCC CRL-1687) were seeded at 20,000 cells per well, with log-phase growth typically reached after 6-18 h.

3. Round 1 : CAR T cell treatment (Day 2)

1. Preparation and seeding of T cells
   1. Prepare the required number of CAR T cells in 100 µL/well in human T cell medium without expansion cytokines (e.g. IL-2, IL-7, IL-15 etc.).
      NOTE: CAR transduction efficiency is measured once at the beginning of the experiment. The desired effector-to-target (E:T) ratio is set according to the number of CAR⁺ T cells relative to the initially plated tumor cells. For example, for a 1:1 E:T ratio with 50 % CAR⁺ T cells and 20,000 BxPC3 tumor cells, 40,000 total T cells should be plated. The E:T ratio therefore refers to the initial condition at the start of the serial killing assay; unless additional measurements are performed, changes in E:T during later rounds remain unknown. Changes in T cell proliferation or survival over time, and hence in the E:T ratio, are considered part of the serial killing readout. If T cells are split between rounds, the E:T is recalculated proportionally (e.g., a 1:1 split after round 3 reduces an initial 2:1 E:T to 1:1 for round 4). If E:T dynamics between rounds are of interest, optional additional wells can be plated for intermediate measurements of CAR % or effector function. Removing anti-CD3/CD28 T cell stimulation beads by magnetic separation can help to reduce unspecific killing. It is also recommended to use T cells in the logarithmic growth phase (typically between 5- and 20-days post-stimulation) and to expand cells with cytokines at least two days prior to the assay, rather than the day before.
   2. Adjust CAR transduction efficiency across experimental groups to the lowest common percentage by mixing with untransduced T cells, based on transduction efficiency analysis.
      NOTE: If all groups have equal transduction efficiency, no adjustment is needed. If CAR⁺ percentages differ, the number of T cells to plate is calculated so that each group starts with the same effective number of CAR⁺ cells and CAR^- cells. This is done by multiplying the desired CAR⁺ cell number (based on the E:T ratio and tumor seeding) by 100 and dividing by the measured CAR⁺ percentage for each group, then adding untransduced T cells if needed to match total T cell numbers. For example, if one group has 50% CAR⁺ T cells, 40,000 T cells are plated (20,000 CAR⁺ + 20,000 CAR^-), while a group with 60% CAR⁺ T cells would plate 33,333 T cells to provide 20,000 CAR⁺ cells, adding 6,667 untransduced T cells to match the total T cell number of the first group (total 40,000).
   3. Optionally, if the cytokine or compound treatment is to be investigated, add 20 µL of a solution consisting of human T cell medium and the desired cytokine or compound at 11x the intended final concentration. This volume is added on top of the standard 200 µL already present in each well (50 µL for blanking, 50 µL for tumor cell seeding, and up to 100 µL for T cell addition). According to the manufacturer, the E-Plate 96 supports a maximum volume of 243 µL ± 5 µL per well, making a total volume of 220 µL acceptable for such optional treatments.
2. T cell seeding and measurement
   1. Remove the E-plate by unlocking the cradle only when ready to add T cells.
   2. Seed prepared T cells directly onto the tumor cells.
   3. Lock the cradle inside the measurement device and abort Step 2 in the RTCA software.
      NOTE: If step 2 is not aborted, the system will continue to measure the previous step and stop only when that step is completed, which results in loss of data points.
   4. Wait for Step 2 to be marked DONE.
   5. Click the PLAY/RUN button to start step 3 (Treatment) and confirm the prompt by clicking OK.
      ​NOTE: Allowing a 15-30 min equilibrium period after handling (e.g., after seeding or transferring cells) before starting the next measurement step (before step 3.2.5) helps minimize initial deflection caused by temperature or pH fluctuations in the growth curves.

4. Round 2 : Tumor cell seeding (Day 3)

1. Prepare a new 96-well xCELLigence E-Plate as described in steps 1.1 and 1.4.
2. Seed fresh tumor cells as described in step 2.
3. Incubate the E-plate overnight to allow cell adherence.

5. Round 2 : CAR T cell transfer to second E-plate (Day 4)

1. Transfer the contents of each well into a corresponding well of a U-bottom 96-well plate using a multichannel pipette. Be careful not to touch the electrodes at the bottom of the E-plate.
2. Centrifuge at room temperature, 400 × g for 5 min.
3. Carefully aspirate the supernatant and retain it for enzyme-linked immunosorbent assay (ELISA) if needed.
4. Resuspend the cell pellet in 100 µL of fresh medium without expansion cytokines.
   NOTE (optional): T cells can now be split (e.g., 50 µL = 1:2 split) and used for further analysis, such as flow cytometry. Splitting T cells adjusts for their proliferation during the xCELLigence killing assay. It helps to reduce the necessary number of cycles until exhaustion becomes apparent. Testing splitting ratios between 2 and 4 is recommended.
5. Add 100 µL/well T cells to the second-round tumor E-plate (50 µL if 1:2 split), ensuring medium and, if applicable, cytokine/compound are fully replenished.

6. Continuation of serial killing assay (Days 5 - 10 and beyond)

1. Repeat Steps 4 and 5 for additional rounds of killing as needed with 48 h intervals.
2. Monitor killing activity and T cell persistence at each stage using xCELLigence readouts and supplementary assays such as ELISA or flow cytometry.

7. Data collection and E-plate handling

1. After the desired measurement duration, press STOP in the software.
2. Export data normalized to the time point of effector-cell addition and non-averaged to enable accurate mean ± SEM plotting in the analysis software (R, GraphPad Prism, etc.). Export the averaged dataset if desired.
3. Unlock and remove the E-plate. Release the experiment in the software.
4. Clean or store the E-plate in the incubator for additional analyses (e.g., supernatant for ELISA or cell harvest for flow cytometry).

8. Optional: E-plate washing for reuse

NOTE: This optional step allows reuse of xCELLigence E-plates to reduce experimental costs. Reuse is only recommended if the electrical properties of the E-plate remain stable across experiments and until individual wells fall within the acceptable baseline range. Follow all safety procedures, especially when handling sodium hydroxide (NaOH). Visually inspect E-plates after washing to detect potential faults in the electrodes.

1. Discard all contents of the E-plate without touching the bottom of the E-plate.
2. Wash each well three times with 200 µL deionized water.
3. Add 200 µL of 0.1 M NaOH to each well.
   CAUTION: NaOH is corrosive. Ensure that local safety guidelines are followed. Wear gloves, coat and safety goggles. Handle 1 M NaOH in a safety cabinet. Discard NaOH waste down the sink with water running to ensure proper dilution. Never mix concentrated acids and bases. Do not add water to acid; only acid to water.
4. Incubate the E-plate at room temperature for 5 min.
5. Wash each well at least three times with deionized water, ensuring all residual NaOH is removed.
6. Dry the E-plate by gently tapping it on a tissue, followed by air drying.
7. Sterilize the E-plate under UV light inside a biosafety cabinet for at least 1 h or a UV irradiation device.
   NOTE: Position the E-plate so that the wells face directly upward. UV light does not penetrate plastic well bottoms.

Successful execution of the protocol yields reproducible impedance-based readouts that reflect tumor cell viability and CAR T cell cytotoxicity over time. Cell growth and cytotoxicity were assessed using the commercial RTCA system. Data were expressed as normalized cell index (NCI), calculated by dividing the CI at each time point by the CI at a selected normalization time. The normalization time is set immediately before the addition of effector cells. Time to clearance is defined as the interval until NCI reaches zero following effector addition. NCI is calculated by the RTCA software and can be exported for further analysis, including killing rate and time to clearance, using statistical software such as GraphPad Prism. Representative NCI curves were generated to visualize cytotoxic kinetics. Further details on calculation principles are available elsewhere25.

In a representative experiment, CAR T cells engineered with a second-generation CAR and an additional chimeric cytokine receptor (CCR) maintained robust lytic potential across multiple rounds of tumor challenge when co-cultured with BxPC-3 pancreatic cancer cells, while CAR T cells without the CCR lost killing capacities after round 02 (Figure 1). Tumor cells were reintroduced every 48 h, and the gradual decline in NCI following each stimulation cycle confirmed the sustained killing capacity of effector cells. In a comparative experiment, two CAR T cell constructs were evaluated for their ability to sustain cytolytic activity over repeated antigen challenges (Figure 2). While one construct exhibited a loss of killing capacity after the fourth stimulation, the other maintained robust tumor clearance through the fifth round, illustrating how the assay distinguishes CAR designs with different persistence profiles.

Conversely, suboptimal outcomes may arise from technical or biological factors. For example, when CAR T cells were administered together with a compound that interfered with impedance measurements, the CI dropped below zero immediately upon addition, producing an artifact that prevented meaningful analysis (Figure 3). Such results highlight the importance of validating potential assay interferences prior to experimental readout. Similarly, when non-adherent tumor cell lines are tested without immobilization strategies, the lack of adhesion prevents CI from rising after seeding, resulting in traces that cannot be used for cytotoxicity analysis.

Together, these examples illustrate the range of outcomes that can occur when applying the protocol and provide benchmarks for distinguishing between successful and suboptimal experiments. Positive readouts in Figure 1 and Figure 2, confirm that CAR T cells are capable of sustained cytotoxic activity in a serial killing setting, whereas negative outcomes, such as shown in Figure 3, indicate technical limitations or inappropriate experimental conditions that require adjustment.

Figure 1. Sustained serial-killing activity of CCR-engineered CAR T cells against BxPC-3 tumor cells. CAR T cells engineered with a chimeric cytokine receptor (CCR) sustained lytic potential after multiple antigen-specific challenges with a BxPC-3 pancreatic cancer cell line, in the presence of relevant CCR-specific cytokine (50 ng/mL). T cells were co-cultured at a 2:1 E:T ratio with BxPC-3 cancer cells. Tumor cells (0.2 x 105) were reintroduced every 48 h after T cell administration. The mean normalized CI of 3 technical replicates is shown (n = 1 experiment). Please click here to view a larger version of this figure.

Figure 2. Comparative persistence of two CAR T cell constructs under repeated antigen challenge. CAR T cells engineered with two different constructs were evaluated for sustained cytolytic potential following repeated challenges with BxPC-3 pancreatic cancer cells. T cells were co-cultured with BxPC-3 cells at an E:T 1:1 ratio. T cells were transferred to freshly plated tumor cells (0.2 × 105) every 48 h. While CAR T construct 1 lost cytolytic activity at the 4th stimulation, construct 2 maintained killing capacity until the 5th round. Data represent the mean normalized cell index (CI) of three technical replicates (n = 1 experiment); shaded areas indicate the standard error of the mean. Please click here to view a larger version of this figure.

Figure 3. Assay interference caused by compound-associated CAR T cell administration. T cells were co-cultured at a 10:1 E:T ratio with BxPC-3 cancer cells (0.2 x 105). The compound interfered with the impedance readout, which immediately dropped below zero upon addition of the T cell-compound mixture. This artifact made the xCELLigence system unsuitable for evaluating this setup. The mean normalized CI of 3 technical replicates is shown (n = 1 experiment). Please click here to view a larger version of this figure.

This protocol describes a method for evaluating CAR T cell-mediated cytotoxicity over multiple rounds of antigen exposure using the xCELLigence RTCA platform. By enabling longitudinal measurement of tumor cell viability via impedance, this approach captures repetitive cytotoxic responses - referred to as serial killing - in a label-free and continuous format. The protocol is designed to monitor how T cell effector function changes over time in response to successive tumor cell re-challenges, providing insight into parameters such as functional persistence, early exhaustion, and overall killing dynamics (Figure 1).

Several procedural aspects are critical to ensuring reproducibility and data quality. First, pipetting in the wells must be performed carefully to avoid damaging the microelectrode surface at the bottom of the well. Second, the blanking step establishes the electrical baseline and identifies any nonfunctional wells before cell addition. Including additional wells during blanking is recommended, as defects such as unstable impedance signals or unusually low or high CI values after blanking (outside the range of 0 - 0.2 CI at baseline) may indicate compromised electrodes that should be excluded from analysis²⁰. Third, tumor cell growth profiles can vary significantly across commonly used cell lines, and adjusting seeding density accordingly (e.g., 5×10³-4×10⁴cells/well) allows flexibility in timing - such as moving from overnight adhesion to same-day T cell co-culture. Forth, T cell seeding and volume planning must adhere to the E-Plate 96's volume range (243 µL ± 5 µL per well at most). While the default configuration uses 200 µL total volume (50 µL blanking, 50 µL tumor cells, 100 µL effector cells), additional treatments can be accommodated by concentrating T cells in 50 µL or adding 20 µL of a 11× cytokine or compound solution. Finally, the culture status of the T cells is important to consider. Besides differences between fresh and frozen cells, engineering procedures such as CAR transduction often lead to strong activation. Sufficient recovery time is essential to lower basal activation levels, particularly when studying the transition from cytotoxicity to exhaustion^26,27.

The protocol can be adapted and modified depending on experimental goals. Optional re-use of E-Plates is included as a cost-reduction step, provided that post-cleaning blanking values remain stable. T cell and co-culture supernatant harvesting can be performed for all rounds with centrifugation in U-bottom plates, and effector cell viability can be monitored when performing multi-round assays. Including appropriate controls-such as target-only, effector-only, and maximum lysis wells (e.g. DMSO)-helps normalize impedance data and interpret killing curves²⁰. Troubleshooting steps are included for common software-related issues, such as interrupted measurements due to failure to abort preceding steps or improper loading of experiment templates.

This protocol requires the simultaneous use of two (plate) positions on the RTCA xCELLigence machine. If access is limited or a multi-plate device is unavailable, the assay can be adapted as follows: step 4 (round 2 - target cell seeding), normally performed on day 3 in parallel with the ongoing round 1 co-culture, is shifted to day 4. After exporting data on day 4, the round 1 plate is removed from the machine and placed in the incubator, freeing up the position to start step 4. On day 5, step 5 (round 2 - effector cell addition) can then be carried out. Notably, overall assay timing can be shortened using the described protocol of morning/afternoon plating instead of consecutive-day steps. Although co-cultures are recorded for 48 h on the device, their actual duration extends to 72 h.

Some limitations of the method should be considered. The impedance readout is based on the adherence of target cells to the electrode surface, making it less suitable for suspension tumor models without additional adaptation²⁸. Commercial reagents for immobilization are available but may alter cell behavior²⁰or invalidate readouts (Figure 2). As a result, this protocol cannot be applied to non-adherent target cells derived from hematological malignancies or adherent effector cells including differentiated CAR-macrophages. Cancer cell lines also vary widely in their culture requirements, growth kinetics (including nutrient consumption), and sensitivity to compounds or effector cells. Therefore, preliminary experiments should be conducted to characterize these properties in the chosen in vitro model before proceeding with killing assays. Furthermore, because CAR T cells are non-adherent, their contribution to impedance is negligible; thus, effector cell proliferation or exhaustion cannot be assessed directly without supplementary assays (e.g., flow cytometry or ELISA). Combining impedance-based functional measurements with multiparametric phenotypic profiling, such as spectral cytometry, can help elucidate mechanisms underlying CAR T cell functional decline²⁹. While plate washing can extend usability³⁰, repeated cleaning can reduce signal sensitivity, and results should be monitored accordingly.

In comparison to conventional cytotoxicity assays - such as chromium release, LDH, or endpoint flow cytometry - the xCELLigence approach allows continuous and label-free monitoring of target cell viability¹⁹. Studies have shown that impedance-derived cytolysis correlates well with traditional apoptosis markers and cell viability measurements^19,24. The ability to track changes in killing dynamics over time, including during repeated tumor challenges, may provide additional resolution in assessing CAR T function beyond static assays.

The described protocol may be useful in preclinical CAR T cell research, particularly when evaluating longitudinal killing kinetics, exhaustion modeling, or effects of external modulators such as cytokines or small molecules. Previous studies have applied similar impedance-based readouts to investigate dose responses, manufacturing conditions, or CAR construct design. This method offers a relatively accessible way to generate kinetic killing data with minimal handling. The optional plate reuse step may also reduce assay costs, which can be a consideration in large-scale experimental setups.