What is Label-Free Viability?
As use of physiologically relevant cell models becomes more common, there is a growing need for label-free, non-perturbing solutions that deliver deep biological insights. Fluorescent labeling prolongs workflows and complicates analysis by introducing an additional variable that may influence study outcomes. Eliminating fluorescent reporters altogether ensures experimental observations are not attributed to the label - or to the labeling process itself.
Benefits of Label-Free Analysis:
- A non-invasive, non-perturbing method for studying the health of cell populations when fluorescent labeling is not feasible, such as with rare cell types
- Rapid advancements in live-cell analysis and computational power in recent years can be adapted to describe how complex analysis is distilled into simple, user-friendly workflows. Artificial intelligence (AI) has provided solutions for real-time, label-free analysis of cell behavior and function
- Label-Free Cell Health Assays enable the study of cells in their normal physiological conditions, a critical step when evaluating new therapeutic drug candidates to treat cancer and other diseases
The Incucyte® Live-Cell Analysis System - when used with the Incucyte® AI Cell Health Analysis Software Module for high-throughput, label-free analysis - helps accelerate workflows while generating data that is reproducible, reliable and free of bias.
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Solutions for Label-Free Viability
Software Overview
Incucyte® AI Cell Health Analysis Software Module
The Incucyte® AI Cell Health Analysis Software Module is an all-in-one analysis tool that segments cells and classifies them as live or dead. It allows users to increase throughput of cell health analyses without need for fluorescent labels, generating accurate and objective data with reduced time requirements and cost.
The Incucyte®️ AI Cell Health Analysis Software Module is available to purchase for all S-series instruments and requires a GPU co-processor (BA-04870) installed as a drop-in hardware upgrade to the controller and 2022B software version. Optional further classification of cell populations based on label-free, or fluorescence parameters (fluorescence intensity within cell boundary) is available in this software module.
Incucyte® AI Cell Health Analysis Workflow
Workflow Figure: With the Incucyte® AI Cell Health Analysis, a neural network (pre-trained with validated datasets) informs segmentation and classification algorithms for accurate processing and quantification of live or dead cells. This AI-driven analysis is applied to all wells and timepoints, providing robust data and visualization of live or dead masks.
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Key Advantages of Label-Free Viability
Use AI-driven algorithms to quantify cell viability kinetically based on trained neural networks
Classify live or dead cells and quantify other biological activity within those populations for greater insight
See example data below
Generate robust and accurate results using diversely trained AI-driven analysis on adherent or non-adherent cell types
See example data below
Example Data for Label-Free Viability
Identify and Count Live and Dead Cells Label-Free - Use AI-driven algorithms to quantify cell viability kinetically based on trained neural networks.
Figure 1: Kinetically monitor and quantify drug-treated adherent and non-adherent cells, label-free. HeLa and Ramos cells were seeded into 96-well plates and treated with either camptothecin or Truxima®, respectively. High-definition (HD) phase-contrast images were acquired every 2h over 2 – 3 days, and cell death was quantified using Incucyte® AI Cell Health Analysis. Phase videos show cell death over time (top) and AI-driven Live (green) and Dead (red) segmentation mask outlines (bottom).
Figure 2: Label-free analysis of cell viability. Ramos B-cell Lymphoma cells were treated with increasing concentrations of anti-CD20 antibody Rituximab and the biosimilar, Truxima®. Cell death was quantified using Incucyte® AI Cell Health Analysis. Timecourse data shows that Rituximab and Truxima® induce time- and concentration-dependent cell death, while toxicity induced by IgG control is minimal. Images display Ramos cells at 48h, either untreated (left) or in the presence of 2 µg/mL Rituximab (right). Untreated cells are healthy and classed as Live (green segmentation) while treated cells show partial cell death and are classified as Dead (red segmentation).
Answer Complex Biological Questions Objectively - Classify live or dead cells and monitor cell development steps within those populations for greater insight.
Figure 3: AI Cell Health read-outs are comparable to standard methods. A549 cells were treated with increasing concentrations of camptothecin. Time-courses represent the % Dead cells quantified using label-free Incucyte® AI Cell Health Analysis (left), with the increase in NIR fluorescence due to the use of Incucyte® Annexin V NIR Dye (middle). Concentration response curves show overlay of these data at 72h (right) and IC50 values are comparable
Figure 4: Analysis of cell cycle markers within live cell subpopulation. HT-1080 cells expressing Incucyte® Cell Cycle Green/Orange Lentivirus were treated with a concentration range of camptothecin (4 nM – 3 µM). Incucyte® AI Cell Health Analysis was performed to identify live versus dead cells and fluorescence classification within the live cell population. Images show the classification masks (purple = live, teal = dead) overlaid with green and orange fluorescence channels. Time-courses show fluorescence classification of the live cell population and reveal a time- and concentration-dependent increase in cells in G1 (orange).
Perform Reproducible, High-Throughput Screening - Generate robust and accurate results using diversely trained AI-driven analysis on adherent or non-adherent cell types.
Figure 5. Incucyte® AI Cell Health in 384-well throughput. Four Cell types were seeded into a 384-well microplate and treated with concentration ranges of camptothecin, cycloheximide and doxorubicin. Microplate view shows the increase in % dead cells over 2 days quantified using Incucyte® AI Cell Health Analysis. Data reveals that camptothecin and doxorubicin induced cell death at high concentrations with comparable IC50 values, while cycloheximide had a cytostatic effect at the concentrations.
Label-Free Viability Technical Resources
Featured Resources
Label-Free Viability Frequently Asked Questions
Yes, in principle. The Caspase-3/7 and Annexin V protocols have been optimized using a range of adherent and non-adherent cell lines and should therefore be applicable to any cell type including cancer cells, immune cells and neurons. Please note some cell types may lack expression of Caspase-3 (e.g. MCF-7 cells). For these cells types we would recommend using the Incucyte® Annexin V Reagents rather than Caspase-3/7 Reagent for apoptosis measurements.
Yes. The protocols and data described here show that it is possible to quantify apoptosis in suspension cell lines such as Jurkat human T lymphocyte cells. You may want to coat your plate to ensure your suspension cells stay in the field of view. Coatings such as poly-L-ornithine, poly-D-lysine or Matrigel™ have been shown to work well.
Yes. As long as the fluorophore is compatible with the Incucyte® Live-Cell Analysis System and does not interfere with the fluorescence emitted by the Incucyte® Caspase3/7 or Annexin V Reagents used.
Yes. The protocols and data described here show that it is possible to quantify two markers of apoptosis (e.g. caspase-3/7 activation and phosphatidylserine externalization) in a single well. For this we recommend that you duplex Incucyte® Annexin V Red Reagent (Cat No. 4641) with the green Incucyte® Caspase-3/7 Reagent (Cat No. 4440).
Yes. Duplex the Incucyte® Caspase-3/7 Reagent (Cat No. 4440) with the Incucyte® Red Cytotox Reagent (Cat No. 4633) or combine the opposing red and green versions of the Incucyte® Annexin V Reagent (Cat No. 4641 or 4642) with Incucyte® Cytotox Reagents (Cat No. 4632 or 4633).
Yes. It is possible to duplex the Incucyte® Annexin V Red Reagent (Cat No. 4641) with Propidium Iodide. We recommend quantifying the Propidium Iodide signal using the red channel of the Incucyte® system and setting a spectral unmixing parameter of 80% Red from Green (please refer to the Fluorescent Dye Optimization Technical Note for more information).***
Data has been generated with both the 10x and 20x objectives. The 10x objective offers the benefit of a greater field of view and reduces the number of images required per well. For higher spatial resolution the 20x objective is recommended.
Incucyte® Apoptosis Assays can be performed using any clear sided or black sided 96 or 384-well clear-bottomed micro-titre plate that is supported on the Incucyte® ZOOM system. We have used Corning 96-well plates (Cat # 3904) for the experiments described here.
No. The Incucyte® Annexin V Reagents have been specially formulated for live-cell imaging with the Incucyte® system. Simply mix-and-read. Washing or fixing is not required.
Yes. There is no requirement to use a specialized assay media or buffer when using Incucyte® Annexin V or Caspase-3/7 Reagents with the Incucyte® system. Integrated Incucyte® image analysis tools enable you to minimize any background fluorescence that may arise from the media, reagents or test agents used. Please do note however that binding of Annexin V to externalized phosphatidylserine is Ca2+-dependent, therefore ensure the Ca2+ concentration in the assay media is ≥1 mM.
We recommended counting apoptotic cells when using the Incucyte® Caspase3/7 Reagent for Apoptosis. The Caspase-3/7 Reagent labels the nuclei of apoptotic cells so it is easy to differentiate between the discretely labelled nuclei of neighboring apoptotic cells. The Incucyte® Annexin V Reagents, however, label the entire cell surface. As such, distinguishing neighboring cells can be challenging. When using Annexin V Reagents we recommend quantifying the total fluorescence area as this is a more robust approach.
Yes, measurements of apoptosis can be normalized to label-free Incucyte® cell confluence measurements. Although some treatments may cause significant cell fragmentation or substantial changes in cell phase contrast we have found the Incucyte® phase contrast confluence metric to be an informative estimate of total cell number over the course of the assay.
It is also possible to normalize to the total number of DNA containing objects at the end of the apoptosis assay. We recommend using Vybrant® DyeCycle™ Green Stain (ThermoFisher) for this purpose. The stain can be added directly to the assay wells without aspiration or washing of the apoptosis reagent containing media.