Liquid tumors such as leukemia, lymphoma, and myeloma are composed of non-adherent cancer cells. These free-floating liquid tumor cells represent an important target in immuno-oncology but assaying suspension cell cultures can be challenging as cells can be easily washed away or disturbed, hindering further measurements. To create more effective strategies for the treatment of blood cancers, accurate assessments of in vitro target-cell killing are critical.
Track liquid tumor cell proliferation in real time
Many hematological cancer cell lines can be analyzed using the Maestro platform. Using an antibody-tethering technique to immobilize the target cells you can monitor cell growth in real time.
Liquid tumor cell lines can be assayed in real time. (Left) Daudi cells on a CytoView-Z 96 plate as imaged by the Omni. (Middle) Proliferation of antibody-tethered vs non-tethered Daudi cells as measured by the Maestro Z shows the increase in signal achieved with antibody-tethering. (Right) The tethering technique is broadly applicable as demonstrated in liquid tumor cell lines Daudi, K562, and Raji across a range of cell densities.
Measure dynamic dose-dependent killing of liquid tumor cells
Real-time assessment can track the magnitude and rate of immune cell-mediated killing. Get a more comprehensive understanding of effector and target cell interactions with kinetic endpoints like kill time 50.
Dose-dependent killing of Daudi cells by CD19-targeted CAR T cells. (Left) Cytolysis of 50K Daudi cells dosed with CD19-targeted CAR T cells at different E:T ratios. (Middle) Kill time 50 (KT50) values for each dose. (Right) A dose-response or immune cell-mediated killing at 96 hours.
Q: Are cell viability dyes required to perform cell proliferation and cytotoxicity assays?
No, cell proliferation and cytotoxicity can be measured label-free with the Maestro Z and Omni platforms.
Q: Why is antibody tethering used for non-adherent cells on the Maestro Z?
Impedance measurements rely on the attachment of non-adherent target cells to electrodes on the bottom of well plates. Tethering antibodies can promote efficient attachment of these cells to the electrodes, hence ensuring an accurate reflection of cellular proliferation.
Read our cell culture protocol to learn more about tethered cell assays in the Maestro Z.
The advantages of measuring immune cell-mediated cell killing on the Maestro Z, ZHT, Pro, and Edge platforms:
Continuous cell monitoring – Up to 384 simultaneous live recordings from your cells. Now you can track immune cell-mediated cell killing in real time, even when you are out of the lab.
Analyze cell activity label-free –Perform noninvasive electrical measurements from the cultured cell population, circumventing the use of dyes/reporters that can perturb your cell model and confound results.
Precise assay environment – No need for an additional cell culture incubator, saving valuable lab space and money. The smart environmental chamber finely controls heat and CO₂ while rejecting electrical noise and mechanical vibrations.
See your cells – Sometimes you just want to look at your cells under a microscope. The Cytoview-Z 96-well plates have a viewing window in each well which allows cell visualization.
Probe cell models in the same plate they were cultured in – other higher throughput platforms (e.g. flow cytometry) often require cell samples to be transferred into a single-cell suspension before testing. In the case of adherent cells this is not ideal since they exist as a functional network of interlinked cells.
Smart phone App for your assay – You can't always be in the lab. But changes in immune cell-mediated cell killing seldom occur at convenient time points. The Maestro Z App allows you to see live results and system status on your smartphone.
It’s easy – With effortless assay setup and intuitive analysis software designed for quick export of figures and results, you can now focus on the science.
Impedance - GeneralShow Full Details
Impedance: For real-time cell analysis
Impedance-based cell analysis is a well-established technique for monitoring the presence, morphology, and behavior of cells in culture. Impedance describes the obstruction to alternating current flow. To measure impedance, small electrical currents are delivered to electrodes embedded in a cell culture substrate. The opposition to current flow from one electrode to another defines the impedance of the cell-electrode interface. When cells are present and attached to the substrate, they block these electrical currents and are detected as an increase in impedance.
Impedance is sensitive to many aspects of cell behavior: attachment, spreading, shape, cell-cell connections (e.g. tight junctions), and death. Even small transient changes, such as swelling or signaling, are detectable by impedance. Because impedance is noninvasive and label free, the dynamics of these changes can be monitored in real time over minutes, hours, or even days without disturbing the biology.
Interdigitated electrodes embedded in the cell culture substrate at the bottom of each well detect small changes in the impedance of current flow caused by cell presence, attachment, and behavior.
In the example below, the electrodes are initially uncovered before cells are added. The electrical current passes easily and the impedance is low. When cells begin to attach and cover the electrodes, less electrical current passes and the impedance is high. After dosing with a cytotoxic agent, cells die or detach, and the impedance decreases back towards baseline.
Impedance measures how much electrical signal (orange arrows) is blocked by the cell-electrode interface. Impedance increases as cells cover the electrode and decreases back to baseline due to cell death.
Continuous cell monitoring
Many cell-based assays are endpoint assays, limited to a single snapshot in time. Repeating these assays at multiple time points can be labor intensive, time consuming, and costly. Key time points can be easily missed. Impedance-based cell analysis is nondestructive and label free, meaning that cellular dynamics can be monitored continuously.
The impedance assay can be used to characterize dynamic cell profiles, revealing how cells grow, attach, and interact over time. Each cell type exhibits a different cell profile, or “fingerprint”, of dynamic cell behavior. These profiles are sensitive to cell type, density, purity, and environmental factors. In this example, the Maestro Z impedance assay readily distinguished cell profiles across different cell densities and cell types.
(A, B) HeLa cells were seeded on a CytoView-Z plate at varying densities and the impedance was continuously monitored by the Maestro Z. Impedance scaled proportionally with cell density and readily distinguished different densities of the same cell type. (C) Maestro monitored the growth of three cell types, HeLa, A549, and Calu-3, and readily distinguishes their distinct cell profiles over time.
The Maestro Z impedance assay can also be used to capture the kinetics of cell responses to drugs or immune cell therapies. The kinetics, which cannot be captured by endpoint assays, often provide key insights into the efficacy of novel therapies. In the example below, the Maestro Z impedance assay was used to quantify the kinetics of cytotoxicity of chemotherapy agents.
A549 cells were dosed with dox, vehicle (DMSO), or tergazyme. Wells dosed with tergazyme showed an immediate decrease in impedance, reflecting complete cell death. Higher doses of dox resulted in a slower decrease in impedance and cell death. Cells dosed with 1 μM dox reached 50% cytolysis at 31 hrs.
Different frequencies reveal cell properties
Impedance varies with frequency, such that different frequencies reveal different aspects of cell biology. The small currents used to measure impedance will always take the path of least resistance. At low frequencies, such as 1 kHz, the impedance of the cell membrane is relatively high, forcing the current to flow under and between the cells. Low frequencies provide details about barrier integrity, the presence of gap junctions, and transepithelial or transendothelial resistance (TEER).
At high frequencies, such as 41.5 kHz, the impedance (and capacitive reactance) of the cell membrane is relatively low. Thus, most of the current couples capacitively through the cell membranes, providing information about the cell layer such as confluency and coverage.
In other words, low frequencies are sensitive to “what” cells are there, whereas high frequencies are sensitive to “how many” cells are there. The Maestro Z impedance assay uses multiple frequencies to provide the most information about the cells, simultaneously, continuously, and in real time.
Multiple frequencies were used to simultaneously and continuously monitor the coverage and barrier function (TEER) of Calu-3 and A549 cells. Coverage, measured as resistance at 41.5 kHz, increases over time for both cell types. TEER, measured at 1 kHz, reveals that only Calu-3 cells form a strong barrier, as they express tight junctions to block flow between neighboring cells.