Cytotoxicity is the quality of being toxic to a cell. Assessing the effect of novel drugs or therapies on in vitro cell viability and health is critical to ensure a treatment is safe and effective. There are many types of cytotoxicity and viability assays available for primary, stem cell-derived, cancer, bacterial, or virus-infected cell culture. When deciding on an assay, some things to consider are:
- Are my dyes or reagents toxic? Do they interfere with the biology I wish to study?
- How do I know which endpoints are needed?
- How complex is the assay and how much hands-on time will it require?
- How much are the reagents, plates, and cells?
The Maestro impedance assay offers a simple solution to those questions. No dyes or complex handling. One plate reveals the full cellular response in one simple assay.
Capture the full time-course of in vitro cytotoxicity and cell viability with a simple live-cell assayAre you working with precious cells, antibodies, patient samples, or compound? Do you have the time required to repeat an assay over multiple time points? Endpoint assays only provide a snapshot of what your cells are doing. To see the full cytotoxic response you need time, money and effort to repeat your experiment over multiple plates and cells. The Maestro impedance assay captures everything on one plate; revealing not only the degree but also the kinetics of cell death. See the Technology tab for more information on how impedance works.
Dose response curves: get the best fit for your data
The cytotoxicity of a treatment depends on the delivered dose. With increasing concentration of doxorubicin, the real-time resistance (left) and cytolysis (middle) curves show increased cytotoxicity. The cytolysis was evaluated as a function of the concentration (right) and fit according to the Hill equation. The analysis estimates the 50% effective concentration (EC50) of doxorubicin to be 0.43 micromolar at 72 hours post-dose.
(Left) SKOV3 cancer cells were cultured at 5k cells per well in a CytoView Z 96-well plate. After 24 hours of culture, the SKOV3 cells were dosed with doxorubicin at 9 concentrations in half-log increments from 0.01 to 100 micromolar. The highest concentrations of doxorubicin produced the lowest measured resistance over time. (Middle) The doxorubicin treated wells were compared with the “No Treatment Control” condition to compute cytolysis. The cytolysis was higher for increasing concentrations of doxorubicin. (Right) The Hill equation was fit to the cytolysis data at 72 hours post-dose. The EC50 in this assay was 0.43 micromolar according to the dose response analysis.
Reliable and robust, Maestro cell viability assays correlate with traditional endpoint assays
The number of cells directly scales with impedance. The correlation is so robust it can meet ISO 20391-2 standards for reproducibility. When compared to a traditional viability endpoint, like an MTT assay, the results directly correlate. In vitro cell viability assays on the Maestro Z, however, are not limited to a single time point.
(Left) Calu-3 cells plated from 6400-51200 cells/well as measured by the Maestro Z. (Middle) 4 hours after plating impedance measurements show a linear relationship that directly correlates with (Right) optical MTT assay results.
Quickly calculate cytolysis and kill time50%
(A) 24 hours after plating, dox is applied and recorded for an additional 36 hours. (B) Dox effect at 30 hours post-dose. (C) Using on-plate controls for cell growth (No Treatment) and death (Tergazyme), the percent cytolysis was tracked in real time throughout the experiment and kill time50 was calculated.
The figures above highlight the dynamic cytotoxic response over time. The percentage of cytolysis, or cell death, can be tracked in real time, and the time to 50% cell death (kill time50) provides insight into the kinetics and efficacy of a treatment's cytotoxicity.
Applications of cytotoxicity and cell viability assays:
CAR-T or other immune cell-mediated killing of cancer cells
Other cancer treatments including chemotherapies and oncolytic viruses
Safety and toxicity testing
Why measure in vitro cytotoxicity and cell viability with Maestro Z?
Analyze the kinetics and capture cytotoxic responses that take minutes to weeks.
Research the biology, not the chemistry. No dyes or reagents to interfere.
More data, less work. One plate for the whole experiment.
Up to 96 wells, save time, cells, media and compound
No complicated assay protocols, just simple cell culture techniques.
Sensitive and reliable. Maestro Z can meet the high reproducibility standards of ISO 20391-2
Getting started with Maestro Z, Pro, and Edge couldn't be easier. Culture your cells in an Axion multiwell CytoView-Z plate (Hour 0). Load this plate into the Maestro system and allow the environmental chamber to automatically equilibrate. Observe cells adhering to the plate and proliferating as changes in the recorded impedance signal (Hour 0 to 24-72). Add test compounds as required. Track changes in cell viability in the CytoView-Z plate label-free and in real-time with the Impedance Module software.
The advantages of measuring cytotoxicity on the Maestro impedance platform:
Continuous cell monitoring – 96 simultaneous live recordings from your cells. Now you can track cytotoxicity 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 cytotoxicity 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.