The ability to differentiate between dead and live cells is essential in the drug discovery process to ensure that potential therapeutic agents are working as intended and do not cause unwanted or unintentional side effects.

Membrane integrity is one of the most common features used to identify dead or dying cells and several assays identify dead cells in this manner. Although these assays also identify live cells by default, through the absence of uptake through the intact membrane, it is more useful and informative to identify live cells specifically with a separate dye. This forms the basis of Live/Dead assays.

Although Live/Dead assays are more commonly applied to cells, variations exist that can differentiate between Live and Dead bacterial and yeast populations with dyes covering a range of fluorescence emission wavelengths.

Live/Dead assay

Live/Dead assays often rely upon both the membrane integrity and either esterase or metabolic activity of live populations and involve the use of two dyes, one that stains live populations, and one that stain dead populations.

The dead cell dye is generally membrane impermeant and therefore cannot pass through intact cell membranes. Once the membrane starts to breakdown and lose its integrity, these dyes can cross freely. Several membrane impermeant dyes are used in these types of assays, including SYTOX, Propidium Iodide (PI) and ethidium homodimer dyes which fluoresce brightly upon binding to DNA. PI, for example, emits a bright red fluorescence (λex 535 nm, λem 617 nm).

The live cell dye is often a substrate for intracellular esterases, such as Calcein AM, which is highly lipophilic, membrane permeant and can pass freely into cells. Once inside cells, Calcein AM is cleaved by intracellular esterases resulting in a fluorescent, membrane impermeant product called Calcein. The Calcein can be detected using fluorescence (λex 495 nm, λem 515 nm) with a microscope. Other dyes that can be used include C12 Resazurin, which identifies metabolic activity in live cells, FUN 1, which identifies metabolic activity in yeast and SYTO 9 which stains all live bacterial and yeast cells regardless of membrane integrity.

Figure 1. Neonatal Dermal Human Fibroblasts, stained with Calcein-AM, imaged on a monochromatic microscope and pseudo-colored (left) from RobertsBiology, Wikimedia comms and live/Dead staining of barium alginate microcapsules containing Sertoili cells, from Biomatlab.fe, Wikimedia comms (Right) both licensed under CC by 2.0.

Live/dead staining dyes are often not compatible with fixation. This has led to the development of several new dyes in Live/Dead fixable staining kits. These are often membrane impermeant dyes that bind to free amine groups on the cell surface and intracellularly, such as LIVE/DEADTM, VivaFixTM and GloCellTM. These dyes bind only to cell surface amines in live cells but can cross the membrane of dead cells to bind intracellular amines, leading to intracellular fluorescence in dead cells.

General assay workflow

The general procedure is as follows:

Figure 2. General process for a high throughput live/dead assay. Treat cells cultured in imaging dishes or plates. Label cells with fluorescence Live/Dead stain and any other required markers. Image and quantify fluorescent signal. Plot results, such as percentage or ratio of red and green fluorescence.
  1. Cell culture and plating on dishes: Cells can be cultured in 2D (monolayers) or 3D (organoids/spheroids) and plated on glass bottomed or other imaging appropriate well plates
  2. Cell treatment: Cells can then be treated with drug candidates or substances of interest
  3. Fluorescent labelling with Live/Dead: Cells are then labelled with Live/Dead dyes, along with any other toxicity markers as required
  4. Measure fluorescence signal: The assay results are generally visualized using fluorescence microscopy, flow cytometry or a plate reader. Automated microscopy imaging can be used to analyze multiple wells in a high throughput manner
  5. Analyze fluorescence: Fluorescence intensity can be quantified to understand live and dead cell populations and to make conclusions regarding the toxicity of the substances being screened / tested. Speed and accuracy can be improved using automated analysis, during or post acquisition, to segment cells (and other features as required), quantify red / dead and green / live fluorescence and determine the ratio of the two.


Live/Dead assays are applicable to a wide range of cell studies as it is essential to ensure cell health during routine experiments. They are also particularly important in drug development and toxicity testing and lend themselves well to high throughput screening techniques.

In toxicity studies, cell models are used that represent potential target organs, such as H4 neuroglioma cells for the brain and HepG2 cells for the liver. Live/Dead assays have been applied to assess the toxicity of a variety of substances, such as nanomaterials, in these types of cell models in a high throughput manner (Chen, 2008).

Some methods make use of more complex 3D models, such as organoids, that better represent the physiological 3D environment. Differentiated HepG organoid models have been used to assess the toxicity of drugs, such as amiodarone and cyclosporine using Live/Dead stains, in combination with other toxicity markers, such as ROS and mitochondrial membrane potential, using high throughput screening methods (LI, et al., 2017).

Similar 3D models have been employed in cancer research. Therapeutic anti-cancer strategies often focus on the induction of cell death in tumor cell populations to shrink tumors. Multi-cellular organoids that represent the 3D tumor environment and Live/Dead cell assays, combined with automated image analysis, have been used to assess in vitro tumor response to chemotherapy drugs such as oxaliplatin, radiation therapy and photo therapy (Bulin, 2017).

Alternative bacterial specific Live/Dead assays are suitable for monitoring viability of bacterial populations. This type of assay can be used to assess bacterial susceptibility to antibiotic treatments (Robertson, 2019).


Cell viability assays, such as the Live/Dead assay are essential for ensuring cell health during in vitro research and drug discovery. These assays can be performed alone, or in conjunction with other toxicity markers and labelling methods to understand the side effects and toxic potential of drugs or treatment protocols. These types of assays lend themselves well to high content imaging and analysis, providing a simple, fast and effective tool that can be applied in the safe generation of new drug therapies.


Biomatlab.fe, n.d. CC BY-SA 4.0 <>, via Wikimedia Commons, s.l.: s.n.

Bulin, A. L. B. M. &. H. T., 2017. Comprehensive high-throughput image analysis for therapeutic efficacy of architecturally complex heterotypic organoids. Scientific reports, 7(1).

Chen, J. Z. J. C. H. H. C. K. L. F. Z. X. R. J. T. W. S. T. C. a. H. X., 2008. Differential cytotoxicity of metal oxide nanoparticles. Journal of Experimental Nanoscience, 3(4), pp. 321-328.

LI, al., 2017. The three dimensional organoids-based high content imaging model for hepatotoxicity assessment. Acta Pharmaceutica Sinica, Volume 12, pp. 1055-1062.

RobertsBiology, n.d. CC BY-SA 4.0 <>, via Wikimedia Commons, s.l.: s.n.

Robertson, J. M. C. V. F. &. S. S. (. O. o. t. P. f. t. L. B. B. V. K. f. R. D. o. B. L. F. i. m. 1. 8. h., 2019. Optimisation of the Protocol for the LIVE/DEAD® BacLightTM Bacterial Viability Kit for Rapid Determination of Bacterial Load.. Frontiers in microbiology, 10(81).