Incucyte® Cytotoxicity Assays for Live-Cell Analysis
Cytotoxicity is a general term that describes the detrimental effects of substances or environmental changes on cell health. Exposure of cells to a cytotoxic stimulus may compromise metabolic activity, inhibit cell growth or division or ultimately produce cell death. 'Necrotic' cell death is a catastrophic cell lysis. 'Apoptotic' cell death is a more controlled, programmed mechanism. 'Autophagy' is a specialized process whereby cells digest themselves from within. Irrespective of the mechanism of cytotoxicity, once a cell irreversibly loses its membrane integrity, it is destined to die.
A number of cytotoxicity assays involve measurement of cell membrane integrity, either with vital dyes that are excluded from healthy cells (e.g. trypan blue or propidium iodide), or via release of markers from dying cells (e.g. cellular proteases). Metabolic activity measurements (e.g. MTT, LDH or ATP assays) are also used to measure cell health and viability. Despite these methods, however, few if any involve direct counting of the number of dying cells over time.
The Incucyte® Cytotoxicity Assay uses Incucyte® Cytotox Dyes to make real-time measurements of cell death based on cell membrane integrity.
Solutions for Cytotoxicity
Incucyte® Cytotoxicity Assay Overview
The Incucyte® Live-Cell Analysis System enables real-time, automated cytotoxicity assays within your tissue culture incubator. The Incucyte® Cytotoxicity Assay uses Incucyte® Cytotox Dyes to make real-time measurements of cell death based on cell membrane integrity.
- Incucyte® Cytotox Green Dye - labels dying cells green
- Incucyte® Cytotox Red Dye - labels dying cells red
- Incucyte® Cytotox NIR Dye – labels dying cells near-IR
When added to the tissue culture growth medium, Incucyte® Cytotox Dyes are inert, non-fluorescent and do not enter viable cells. As cells die and membrane integrity is lost, the Incucyte® Cytotox Dye enters the cell and fluorescently labels the nuclei. Dying cells are identified and quantified over time by the appearance of green/red/NIR labeled nuclei. High-definition phase contrast images and movies provide an additional validation of cell death based on morphology (e.g. loss of cytoskeleton structure, loss of motility).
Incucyte® Cytotoxicity Assay Concept
- Detect cytotoxicity in real time by adding mix-and-read Incucyte® Cytotox Dyes to your cultures
- Automatically quantify number of dying cells using intuitive Incucyte® live-cell imaging and analysis tools
- Multiplex cytotoxicity readouts with measurements of proliferation or apoptosis by combining with Incucyte® Nuclight nuclear labeling reagents, Incucyte® Caspase 3/7 Dye or Incucyte® Annexin V Dye
Measure cytotoxicity in tumor, immune or neuronal cultures using the Incucyte® Cytotoxicity Assay.
(Left) Real-time detection of cytotoxicity in HT-1080 fibrosarcoma cells following treatment with the cytotoxic drug camptothecin. Dying cells are labeled green, in real time, by the mix-and-read Incucyte® Cytotox Green Dye
(Right) Incucyte® Cytotox Red Dye labels dead rat forebrain neurons treated with 333 μM glutamate.
Incucyte® Cytotox Dyes are fully validated for use with the Incucyte® Live-Cell Analysis System and cytotoxicity assays. Furthermore, they can be combined with our range of Incucyte® Nuclight nuclear labeling reagents, the Incucyte® Caspase 3/7 Dye, or the Incucyte® Annexin V Dye for multiplexed measurements of proliferation and apoptosis alongside cytotoxicity in a single well.
Cytotoxicity Assays with the Incucyte® Live-Cell Analysis System
To kinetically measure cell membrane integrity, live-cell analysis methods have been developed. However, optimized technology and reagents are required for flexible and accurate assessment. Use of the Incucyte® Live-Cell Analysis System in conjunction with Incucyte® Cytotox Dyes, as a live-cell kinetic assay for the measurement of cytotoxicity, demonstrates quantitative and reproducible detection of cell permeability - a hallmark of cell death.
This approach also provides the ability to monitor morphological changes in parallel with quantification, in physiologically relevant conditions. The combination of Incucyte® Cytotox Dyes with Incucyte® Nuclight Reagents or cells lines enables multiplexed measurements of proliferation and cytotoxicity.
Key Advantages of Incucyte® Cytotoxicity Assay
Monitor cytotoxicity in your cells by using Incucyte® Cytotoxicity Assays to capture and quantify cells undergoing cell death in real time.
See example data below
Time-lapse imaging of cells and automated data analysis make it simple and easy to determine how and when treatment effects occurred.
See example data below
Incucyte® Cytotox Dyes are non-perturbing and highly sensitive dyes suited for a simple mix-and-read, real-time quantification of cell death.
See example data below
Perform multiplexed measurements of proliferation and apoptosis using Incucyte® Nuclight nuclear labelling reagents, Caspase or Annexin Dyes.
See example data below
Example Data for Cytotoxicity Assay
Visualize and quantify cytotoxicity using time-lapse imaging
Observe cell death over time and measure cytotoxicity using intuitive Incucyte® integrated image analysis tools. Validate treatment effects with images and movies.
Figure 1. Real-Time Visualization and Quantification of Non-Adherent Jurkat Cell Death in Response to the Anti-Cancer Drug Camptothecin (CMP) Using the Incucyte® Live-Cell Analysis System. Jurkat cells (20,000 cells/well) were treated with 3 µM CMP in the presence of Incucyte® Cytotox NIR Dye. Images were acquired every 2 h at 20X for the duration of the experiment. Representative phase-contrast and blended fluorescence images (NIR pseudo-colored blue; top row) and the segmentation mask generated using Incucyte® integrated image analysis tools (red; bottom row) are shown over time (0–24 h). Observe increase of nucleic acid binding by Incucyte® Cytotox NIR Dye as measured by NIR Object Count (8 at 0 h vs. 556 Objects/Image at 24 h). The fluorescent signal from the Incucyte® Cytotox Dyes can be correlated with morphological changes associated with cell death.
Automatic analysis of the time course of cell death within your incubator
Determine how and when treatment effects occurred without removing cells from the stable environment of the incubator - ideal for long-term studies (0 to <10 days).
Figure 2. Quantify treatment effects automatically and non-invasively. Incucyte® Cytotoxicity Assay allows every well of a 96/384 well plate to be imaged and analyzed automatically to provide a microplate readout of cytotoxicity over time (top). Time-courses reveal concentration-dependent treatment effects (bottom left). Transform data into concentration-response curves to compare pharmacology (bottom right).
Simple mix-and-read 96/384-well protocols - no washing, no fixing, no lifting
Plate cells, add your treatments along with the Incucyte® Cytotoxicity Assay and Incucyte® Cytotox Dye and read kinetically in the Incucyte® Live-Cell Analysis System. Read up to 6 x 384-well plates at once for medium/high-throughput screening.
Figure 3. Adherent cell line protocol for the Incucyte® Cytotoxicity Assay.
Multiplex with proliferation and apoptosis measurements
Combine Incucyte® Cytotoxicity Assays with Incucyte® Nuclight nuclear labeling reagents, Caspase-3/7 Dyes, or Annexin V Dyes for multiplexed measurements of proliferation/apoptosis. Readily discriminate between cytotoxic and cytostatic treatment effects.
Capture proliferation label-free, using Incucyte® Cell-by-Cell Analysis Software Module, thus enabling individual cell segmentation and classification based on fluorescence. Calculate the cytotoxic or apoptotic index within the Incucyte® software.
Figure 4. Multiplex cytotoxicity measurements with live-cell counting. Camptothecin (150 nM) treated HT-1080 fibrosarcoma cells (labeled with Incucyte® Nuclight Red Lentivirus) in the presence of the Incucyte® Cytotox Green Dye to detect live/dead cells over time.
Figure 5. Multiplex cytotoxicity measurements with live-cell label free counting and quantification of time-courses and concentration-dependence of cytotoxicity and proliferation. Camptothecin (1 µM) or cycloheximide (1 µM) treated HT-1080 fibrosarcoma cells in the presence of the Incucyte® Cytotox Green Dye to detect live/dead cells at 24h (masking of dead cells shown). Classification plots to identify green (dead) population (second column), time-course plots (third column) and concentration-response curves (forth column) show difference in effect of a cytotoxic and cytostatic compound.
Cytotoxicity Assay Technical Resources
Reagents-Consumables-Brochure-en-L-8000-0738-B00-Sartorius.pdfPDF | 6.1 MB
Incucyte® Cytotox DyesPDF | 501.1 KB
The Incucyte® Live-Cell Analysis System enables real-time, automated quantification of cell proliferation assays inside your tissue culture incubator by: (1) label-free, confluence, (2) label-free, direct cell count, (3) fluorescent labelling, direct cell count.
The Incucyte® Live-Cell Analysis System enables real-time, automated apoptosis assays inside your tissue culture incubator. Measure multiple apoptotic pathways simultaneously and in real time using the mix-and-read Incucyte® Caspase-3/7 and Annexin V Dyes.
Visualize, quantify and understand the dynamic interactions of immune and cancer cells with the Incucyte® Immune Cell Killing Assays.
Incucyte® 3D Tumor Spheroid Assays offer an integrated turnkey solution to automatically monitor and quantify tumor spheroid formation, growth and health in real time inside your tissue culture incubator.
Cytotoxicity Frequently Asked Questions
I study cell death in the tumor microenvironment. Can cell proliferation and cell death assays be multiplexed for the study of cell fate in complex tissues?
Yes. The interaction of cells in complex, tissue-like relationships can be studied in vitro with co-cultures, and cell death can be tracked in those co-cultures with markers of apoptosis and membrane integrity. For the most meaningful data, cell proliferation and cell death can be monitored at the same time in the same culture, with live-cell imaging.
To track a specific cell type throughout the co-culture experiment, start by transducing the cells with one of the NucLight reagents, ensuring that it is a different color than the Cytotox reagent used. The cells will grow and associate into dense, interacting cultures, and cell death will be monitored with the Cytotox or Caspase 3/7 reagents.
Note: All dead and dying cells will be positive for the Cytotox or Caspase 3/7 reagent, not just the ones transduced with the NucLight reagent.
The most commonly used method for predicting cell death in cytotoxicity assays is by using a reagent that is impermeant to intact lipid bilayers. Upon the onset of the cell death cascade, the lipid bilayer becomes porous and leaky, allowing the reagent into the cells where it stains the nucleus. Other assays are capable of detecting different stages and forms of cell death. Apoptosis assays detect the presence of certain markers of dying cells, like activated caspases and Annexin V.
The three assays you mentioned are not mutually exclusive. Cytotoxicity (or cytotox) assays could easily be measured alongside cell proliferation or cell migration assays. The only factors to keep in mind are that you'll need to have enough imaging channels to monitor the readouts from all of the assays at the same time. For live-cell imaging with the Incucyte® that means you could track cell confluence (which needs no fluorochrome), chemotaxis (in either the red or green channel), and cytotoxicity (in the remaining channel).
Electronic pipettes reduce human variance as they take care of aspiration and dispensing. Their multiple pipetting modes speed up work. Also, electronic pipettes offer security features, like calibration reminders and password protection for saved programs.
How sensitive are cytotoxicity assays? Can they differentiate between a population of cells undergoing apoptosis and a single cell lysing, and spilling its contents?
The range of cytotoxicity assays is very broad, so it's impossible to provide an answer that applies to all cytotoxicity assay methods. However, for methods that involve a visual-recognition component alongside a fluorescent intensity component, it is possible to differentiate between a number of cells undergoing apoptosis and a single cell lysing. Specific apoptosis assays, when multiplexed, can even provide finer detail, including signaling the onset of the apoptotic cascade or the derangement of the lipid membrane. Assays like ELISAs, where signal is entirely cumulative, could not provide details like these.
My cell death analysis curves are not reliable, and they don’t have the expected shape. Am I running this cytotoxicity assay incorrectly?
A common reason for this problem is continued cell growth throughout the cell death assay, leading to overcrowding in the well. When plating your cells for cytotoxicity assays, make sure to have an exact count, and don't seed so many cells that your cells will become contact inhibited (by reaching 100% confluence) before your endpoint.
When running a dose-response cytotoxicity assay, wells containing a low concentration of my cytotoxic reagent have a higher proliferation rate than my control and high-concentration wells. Shouldn’t my cell death curve reflect lower cytotoxicity in the presence of less cytotoxic agent?
Occasionally, cytotoxic substances can have the paradoxical effect of stimulating cell proliferation at sublethal concentrations. If you routinely see this effect with your combination of cells and cytotoxic agent, this may be the explanation.