Common approaches to measure a viral titer often rely on the quantification of cytopathic effect (CPE) in plaque assays or the calculation of the 50% tissue culture infectious dose, known as TCID50. However, achieving high levels of accuracy and reproducibility with these traditional methods can be challenging.
Axion BioSystems' Maestro Z, Pro, and Edge systems offer impedance-based cell analysis for real-time, continuous, label-free monitoring of CPEs. Continuous data reveals the kinetics of in vitro viral infections for better understanding of viral CPEs without the time- and cost-intensive process of repeating multiple endpoint assays.
Calculate viral titers label free and in real time
COVID-19 is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2, SARS-CoV-2. While early symptoms may present as relatively mild, they can develop into acute respiratory distress syndrome (ARDS) as the virus damages and disrupts healthy lung tissue. Axion BioSystems' Maestro impedance platform makes it quick and easy to monitor the full time course of viral cell killing.
Vero E6 cells were seeded into the CytoView-Z 96-well plate and impedance was continuously monitored on the Maestro Z system. After 96 hours, different concentrations of SARS-CoV-2 virus were added to determine the lowest multiplicity of infection (MOI) that elicits cell death. (A) The full time course of cell death in response to varying amounts of SARS-CoV-2. (B) Bar graph of impedance at 130 hour timepoint (34 hours after treatment). The addition of the virus resulted in increased cell death in infected cells that correlated with increasing virus concentrations. Uninfected wells continued to show high impedance values, which indicates high cell viability. Data courtesy of Drs. Alex Jureka and Chris Basler at Georgia State University.
Getting started with Maestro Z, Pro, and Edge couldn't be easier. Culture your cells in an Axion multiwell CytoView-Z plate (Day 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 24-96). Add virus and antivirals, and track cytopathic effects (CPE) label free and in real time with the Impedance Module software.
The advantages of measuring viral titer on the Maestro Z, Pro, and Edge platforms:
Continuous cell monitoring – 96 simultaneous live recordings from your cells. Unlike plaque assays, now you can track cytopathic effects (CPEs) in real-time, even when you are out of the lab.
Analyze cell activity label free –The systems 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 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.
Smart phone App for your assay – You can't always be in the lab, but cytopathic effects (CPEs) seldom occur at convenient time points. The Maestro Z App allows you to see live results and system status.
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 doxorubicin, vehicle (DMSO), or tergazyme. Wells dosed with tergazyme showed an immediate decrease in impedance, reflecting complete cell death. Higher doses of doxorubicin resulted in a slower decrease in impedance and cell death. Cells dosed with 1 μM doxorubicin 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.