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Cell Viability AssaysReagents, assays, and kits to evaluate cellular viability using microscopy, flow cytometry, or microplate readers |
Overview of cell viability assays
Cell viability refers to the number of live, healthy cells in a sample [1]. Cell viability assays are used to measure the physical and physiological health of cells in response to extracellular stimuli, chemical agents, or therapeutic treatments [1–3], or when determining optimal growth conditions in cell culture.
Thermo Fisher Scientific offers a vast array of assays for detecting cell viability. Most assays can be used across multiple detection platforms including fluorescence microscopy, flow cytometry, and microplate readers. Each assay contains a specially designed reagent that determines viability based on cellular membrane integrity, cellular function such as enzymatic activity, or metabolic activity. Each viability reagent provides a single-parameter readout on whether cells are living or dead. Cell viability kits are also available and provide the ability to detect multiple measures of cell health. These viability kits allow for simultaneous detection of live, dead, and damaged/dying cells. Cell viability kits help provide more context of cellular changes than a single-parameter readout.
Selection guide of cell viability assays
Nucleic acid binding dyes are cell-impermeant, nucleic acid-binding stains that are only able to enter cells with a compromised plasma membrane. They are dimly fluorescent in aqueous medium, but they highly fluoresce when bound to double-stranded DNA or RNA.
Learn more about these membrane integrity dyes
| Platform* | Standard filter set | Laser (nm) | Ex/Em** (nm) | Cat. No. | |
|---|---|---|---|---|---|
| Propidium iodide (PI) | FC, FM | RFP | 488/532/561 | 535/617 | P1304MP |
| TO-PRO-3 Iodide | FC, FM | Cy5 | 633 | 642/661 | T3605 |
| 7-AAD | FC, FM | Texas Red | 561 | 546/647 | A1310 |
| SYTOX Blue Nucleic Acid Stain | FC, FM | BFP | 405 | 444/480 | S11348 |
| SYTOX Green Nucleic Acid Stain | FC, FM | GFP | 488 | 483/503 | S7020 |
| SYTOX Orange Nucleic Acid Stain | FC, FM | RFP | 561 | 547/570 | S11368 |
| SYTOX Deep Red Nucleic Acid Stain | FC, FM, M | Cy5 | 633 | 660/682 | S11381 |
| SYTOX Blue Dead Cell Stain | FC, FM | - | 405 | 444/480 | S34857 |
| SYTOX Green Dead Cell Stain | FC, FM | - | 488 | 504/523 | S34860 |
| SYTOX Orange Dead Cell Stain | FC, FM | - | 561 | 547/570 | S34861 |
| SYTOX AADvanced Dead Cell Stain Kit | FC | - | 561 | 546/647 | S10349 |
| SYTOX Red Dead Cell Stain | FC, FM | - | 633 | 640/658 | S34859 |
| SYTOX Dead Cell Stain sampler pack | FC, FM | - | 488, 532, 561, 633 | multiple | S34862 |
| *FC = flow cytometry; FM = fluorescence microscopy; M = microplate assay **Ex = excitation; Em = emission | |||||
Membrane integrity viability dyes (nucleic acid binding)
Membrane integrity is a commonly measured parameter in cell viability assays. Membrane integrity dyes are cell-impermeant and only able to enter cells with a compromised plasma membrane. They are not able to penetrate the uncompromised plasma membrane of live cells. Membrane integrity viability dyes comprise:
- Nucleic acid-binding dyes (this section): These viability dyes are non-fluorescent in aqueous media but exhibit increased fluorescence upon binding to double-stranded DNA (dsDNA) or RNA. Nucleic-acid viability dyes include SYTOX dead cell stains, propidium iodide (PI), and 7-aminoactinomycin D (7-AAD).
- Amine-reactive dyes (next section: Membrane integrity viability dyes (amine-reactive)): These viability dyes covalently bind to the primary amine groups available on cellular proteins that are located on the exterior and interior of cells. When bound to surface amine proteins only, the resulting signal is dim; however, when the dye enters cells with a compromised plasma membrane and reacts with interior amine cellular proteins, the resulting signal is highly intense. Amine-reactive viability dyes include the LIVE/DEAD Fixable Dead Cell Stains for flow cytometry.
PI and 7-AAD
PI and 7-AAD are known as classic DNA-binding dyes commonly used in flow cytometry; however, both can also be used in fluorescence microscopy.
PI is a nuclear and chromosome stain commonly used to detect dead cells. In aqueous solution, PI has an excitation/emission spectra of 493/636; however, upon binding DNA, its fluorescence is enhanced 20- to 30-fold to an excitation/emission maximum of 535/617. This dye is commonly used in combination with apoptosis dyes such as annexin V; more information can be found in the Cell Viability Kits section below. With the addition of RNase, PI can also be used for DNA content cell cycle analysis in fixed cells (Figure 1).
Learn more: Cell cycle analysis assays
Figure 1. Detection of proliferation in Wil2S Lymphoma B cells. Cells were treated with 10 µM 5-bromo-2´-deoxyuridine (BrdU) in culture medium for one hour, then pelleted and fixed with cold 70% ethanol. After treatment with RNase and 4 M HCl (to denature the DNA), the cells were labeled with anti-BrdU and detected using green-fluorescent Alexa Fluor 488 Goat Anti–Mouse IgG antibody. In addition, the cells were labeled with red-fluorescent propidium iodide to assess the total cellular DNA content. The cells were analyzed by flow cytometry using 488 nm excitation; the fluorescent signals were collected at ~525 nm for the Alexa Fluor 488 dye and at ~675 nm for propidium iodide. Increased BrdU incorporation is indicative of actively proliferating cells.
7-AAD is another nuclear and chromatin dye that undergoes a spectral shift after binding to DNA (Figure 2). It selectively binds to GC regions of DNA resulting in a distinct banding pattern which allows for its use in chromosome banding studies. 7-AAD can also be used in cell cycle analysis for flow cytometry, and in cells that have fixed and permeabilized.
Figure 2. Wheat root tips in seven stages of the cell cycle. 7-aminoactinomycin D. Panel of confocal micrographs showing cells from wheat root tips in seven stages of the cell cycle. DNA was stained with 7-aminoactinomycin D, and microtubules were labeled with an anti-ß-tubulin antibody in conjunction with a fluorescein-labeled secondary antibody. Cells vary in width from about 15 µm to about 25 µm. The stages are (from top left): interphase cortical microtubule array; pre-prophase band of microtubules (predicts future plane of division); metaphase mitotic spindle; telophase, showing early phragmoplast and cell plate; fully developed phragmoplast during cytokinesis; late cytokinesis (plane of division matching plane of earlier pre-prophase band); restoration of cortical arrays in daughter cells. Image contributed by B.E.S. Gunning, Plant Cell Biology Group, Research School of Biological Sciences, Australian National University. Used with permission from Gunning, B.E.S. and Steer, M.W., Plant Cell Biology: Structure and Function, Jones and Bartlett Publishers (1995).
SYTOX Dead Cell Stains
SYTOX dead cell stains are easy-to-use, cell-impermeant dyes with increased fluorescence upon binding to dsDNA. These viability dyes can be used in cells without an additional wash step and visualized with minimal background staining since they are non-fluorescent in aqueous media. Available in multiple single-color formats and compatible with multiple lasers, these dyes allow the flexibility needed for multiplex experiments and across different platforms including fluorescence microscopy (Figure 3), flow cytometry (Figure 4), and microplates.
Learn more: Nucleus/nucleolus structure dyes
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Figure 3. Bovine pulmonary artery endothelial cells (BPAEC). MitoTracker Red CMXRos, SYTOX Green nucleic acid stain, biotin-XX goat anti–mouse IgG antibody and Cascade Blue NeutrAvidin biotin-binding protein. Bovine pulmonary artery endothelial cells (BPAEC) incubated with the fixable, mitochondrion-selective MitoTracker Red CMXRos. After staining, the cells were formaldehyde-fixed, acetone-permeabilized, treated with DNase-free RNase and counterstained using SYTOX Green nucleic acid stain. Microtubules were labeled with a mouse monoclonal anti–ß-tubulin antibody, biotin-XX goat anti–mouse IgG antibody and Cascade Blue NeutrAvidin biotin-binding protein. This photograph was taken using multiple exposures through bandpass optical filters appropriate for Texas Red dye, fluorescein and DAPI using a Nikon Labophot 2 microscope equipped with a Quadfluor epi-illumination system.
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Figure 4. Viable cell gating with SYTOX Dead Cell Stains. A mixture of heat-treated and untreated human peripheral blood leukocytes was stained with Invitrogen Alexa Fluor 488 dye–conjugated anti–human CD3 antibody, R-PE–conjugated anti–human CD8 antibody, and 5 nM SYTOX Red Dead Cell Stain before analysis by flow cytometry using 488 nm and 635 nm excitation. (A) Histogram showing distribution of two cell populations—dead cells that exhibit significant red fluorescence signal, and live cells, which do not. (B) Dual-parameter plot of CD8 and CD3 staining after gating on live cells.
Membrane integrity viability dyes (amine-reactive)
Amine-reactive viability dyes are cell-impermeant and react with cellular amine proteins. On live cells, these dyes bind to extracellular primary amines and dimly fluoresce; in dead cells, they bind to intracellular amine proteins resulting in a significant increase in cellular fluorescence. Since the binding to cellular proteins is covalent, the samples can be fixed and permeabilized without losing their viability staining pattern.
Image-iT DEAD Green
Image-iT DEAD Green (Figure 5) is another easy-to-use cell-impermeant, membrane integrity dye used to identify dead cells. This viability stain can be used in cells that will undergo fixation and/or permeabilization and allows for multiplex experiments with other fluorescent dyes.
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Figure 5. Single-channel viability assay with multiplexing. HeLa cells treated with camptothecin and stained with Hoechst 33342 (blue channel) for a total cell count and dead cells are stained with Image-iT DEAD Green (green channel).
Amine-reactive viability dyes are cell-impermeant and react with cellular amine proteins. Dimly fluorescent when bound to extracellular amine proteins on live cells, there is a significant increase in fluorescence when these dyes enter dead cells and bind to intracellular amine proteins. Since this binding is covalent, these samples can be fixed and permeabilized without losing their viability staining pattern.
Learn more about these fixable viability dyes
| Platform* | Standard filter set | Laser (nm) | Ex/Em** (nm) | Cat. No. | |
|---|---|---|---|---|---|
| Image-iT DEAD Green Viability Stain | FC, FM | GFP | 488 | 488/515 | I10291 |
| LIVE/DEAD Fixable Blue Stain | FC | - | UV | 350/450 | L34961 |
| LIVE/DEAD Fixable Violet Stain | FC | - | 405 | 416/451 | L34963 |
| LIVE/DEAD Fixable Lime Stain | FC | - | 405 | 405/506 | L34989 |
| LIVE/DEAD Fixable Aqua Stain | FC | - | 405 | 367/526 | L34965 |
| LIVE/DEAD Fixable Yellow Stain | FC | - | 405 | 400/575 | L34967 |
| LIVE/DEAD Fixable Green Stain | FC | - | 488 | 495/520 | L34969 |
| LIVE/DEAD Fixable Olive Stain | FC | - | 488 | 479/557 | L34977 |
| LIVE/DEAD Fixable Orange Stain | FC | - | 561 | 578/602 | L34983 |
| LIVE/DEAD Fixable Red Stain | FC | - | 488, 561 | 595/615 | L34971 |
| LIVE/DEAD Fixable Far Red Stain | FC | - | 633 | 650/665 | L34973 |
| LIVE/DEAD Fixable Scarlet Stain | FC | - | 633 | 702/723 | L34986 |
| LIVE/DEAD Fixable Near-IR (775) Stain | FC | - | 633 | 750/775 | L34975 |
| LIVE/DEAD Fixable Near-IR (780) Stain | FC | - | 633 | 633/785 | L34992 |
| LIVE/DEAD Fixable Near IR (876) Stain | FC | - | 808 | 840/876 | L34980 |
| *FC = flow cytometry; FM = fluorescence microscopy **Ex = excitation; Em = emission | |||||
LIVE/DEAD Fixable Dead Cell Stains
LIVE/DEAD Fixable Dead Cell Stains are cell-impermeant, amine-reactive dead cell stains optimal for use in cells that will undergo fixation and/or permeabilization. These stains are used exclusively for determining cell viability in flow cytometry (Figure 6). Available in multiple single-color formats and compatible with multiple lasers, these dyes provide the flexibility needed for multiplex experiments.
Learn more about Fixable Viability Dyes for Flow Cytometry
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Figure 6. Retention of LIVE/DEAD Fixable Viability Stains after fixation. The LIVE/DEAD Fixable Red (615) Viability kit for 488 and 561 nm excitation was used to differentially stain a mixture of live (left peak) and heat-treated Jurkat cells (right peak). Cells in (A) were not fixed; cells in (B) were fixed in 3.7% formaldehyde following staining. Samples were analyzed by flow cytometry using 488 nm excitation and ~615 nm emission.
Enzyme activity viability substrates are nonfluorescent reagents that react with cellular enzymes in live cells (CellTrace Calcein AM dyes) or react with cellular enzymes released from damaged cells (CyQUANT Cytotoxicity Assays) and use a fluorescent or colorimetric detection method.
Learn more about these enzyme activity substrates.
| Name | Platform* | Detection method | Absorbance (nm) | Fluorescence Ex/Em** (nm) | Cat. No. |
|---|---|---|---|---|---|
| CellTrace Calcein Blue, AM | FC, FM | Fluorescence | - | 323/439 | C34853 |
| CellTrace Calcein Violet, AM | FC, FM | Fluorescence | - | 400/452 | C34858 |
| CellTrace Calcein Green, AM | FC, FM | Fluorescence | - | 495/515 | C34852 |
| CellTrace Calcein Red-Orange, AM | FC, FM | Fluorescence | - | 577/590 | C34851 |
| Calcein, AM | FM | Fluorescence | - | 494/517 | C3100MP |
| CyQUANT LDH Cytotoxicity Assay - Fluorescence | M | Fluorescence | - | 560/590 | C20302 |
| CyQUANT Cytotoxicity Assay Kit (G6PD Release Assay) | M | Colorimetric, fluorescence | 563 | 560/590 | V23111 |
| CyQUANT LDH Cytotoxicity Assay | M | Colorimetric | 490 | - | C20300 |
| *FC = flow cytometry; FM = fluorescence microscopy; M = microplate assay **Ex = excitation; Em = emission | |||||
Enzymatic activity substrates
Enzymatic activity substrates are fluorogenic reagents that are non-fluorescent and either react with cellular enzymes in live cells or cellular enzymes released from damaged cells to become fluorescent or colorimetric. Enzymatic activity substrates comprise:
- CellTrace Calcein AM dyes: These viability dyes are cell-permeant, fluorogenic substrates that react with intracellular enzymes in live cells to become fluorescent. Within live cells, the AM group is cleaved by active esterases, resulting in fluorescence. This AM group cannot be cleaved by dead cells. Calcein AM dyes are ideal for short-term staining since the dyes can be transported out of the cell within a few hours.
- CyQUANT cytotoxicity assays: These viability assays detect extracellular glucose-6-phosphate dehydrogenase (G6PD) or lactate dehydrogenase (LDH) released from damaged cells. CyQUANT Cytotoxicity Assay Kit (G6PD Release Assay) and CyQUANT LDH Cytotoxicity Assay detect G6PD or LDH, respectively, by an enzymatic process that leads to the reduction of resazurin into red-fluorescent resorufin. CyQUANT LDH Cytotoxicity Assay is a colorimetric assay that detects LDH by an enzymatic process that leads to the formation of a red formazan product.
Learn more: CyQUANT cytotoxicity assays
CellTrace Calcein AM
CellTrace Calcein AM dyes are cell-permeant substrates that identify live cells. These substrates measure both enzymatic activity and membrane integrity. The enzymatic activity of live cells allows the AM group to be cleaved by esterases resulting in fluorescence, while the intact cell membrane of live cells is required for the intracellular retention of the fluorescent dye. Available with blue, violet, and green fluorescence, these dyes facilitate the flexibility you need for multiplex experiments and can be used with fluorescence or confocal microscopy (Figure 7) or in flow cytometry experiments (Figure 8).
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Figure 7. Primary Rat Cortex Neurons (A1084001) were treated with Calcein AM and with the FluxOR Red reagent. After stimulation of the potassium channels, cell viability was determined by imaging with a FITC/Alexa Fluor 488 filter setting to detect the green emission from Calcein AM (top left panel). The potassium ion flux was detected using FluxOR Red and a TRITC/Alexa Fluor 555 filter set (top right panel). The overlay of Calcein AM and FluxOR Red confirms cell viability and potassium ion flux (bottom panel).
Figure 8. Calcein AM viability dye. BALB/c thymocytes were stained with 12.5 nM Calcein AM for 30 minutes at room temperature (left). Thymocytes were kept on ice overnight (shaded histogram) or cultured overnight at 37°C without (purple) or with (blue) 1 µM dexamethasone. Thymocytes cultured overnight without dexamethasone were also stained with 7-AAD allowing further discrimination between live and dead cells (right). Total cells were used for analysis.
Cell viability assays using esterase substrates and amine-reactive dyes
The LIVE/DEAD Violet Viability/Vitality Kit provides a two-color fluorescence assay based on the measure of two essential cell health parameters: plasma membrane integrity to measure dead cells and intracellular esterase activity to measure live cells (Figure 9). CellTrace Calcein Violet AM and LIVE/DEAD Fixable Aqua fluorescent reactive dye are optimal for this application as both stains utilize the violet laser, allowing other laser lines to be used with other fluorescent dyes.
Figure 9. Staining of a mixture of heat-killed and untreated Jurkat cells. Jurkat cells were stained according to the protocol in the LIVE/DEAD Violet Viability/Vitality Kit. Cells were analyzed using a flow cytometer equipped with a 405 nm laser and a 450/50 nm bandpass filter for calcein violet–labeled live cells (L), and a 525/50 nm bandpass filter for the aqua dye–labeled dead cells (D).
Metabolic activity viability assays are live-cell, membrane permeant reagents that detect the cellular reducing environment (alamarBlue, PrestoBlue) or the cellular redox potential (CyQUANT MTT or CyQUANT XTT) and use a fluorescent or colorimetric detection method.
Learn more about these metabolic activity reagents.
| Name | Platform* | Detection method | Absorbance (nm) | Fluorescent Ex/Em** (nm) | Cat. No. |
|---|---|---|---|---|---|
| alamarBlue Cell Viability Reagent | M | Fluorescence | 570 | 560/590 | DAL1025 |
| alamarBlue HS Cell Viability Reagent | M | Fluorescence | 570 | 560/590 | A50100 |
| PrestoBlue Cell Viability Reagent | M | Fluorescence | 570 | 560/590 | A13261 |
| PrestoBlue HS Cell Viability Reagent | M | Fluorescence | 570 | 560/590 | P50200 |
| Vybrant Cell Metabolic Assay Kit, with C12-resazurin | FC, M | Fluorescence | 563 | 563/587 | V23110 |
| CyQUANT MTT Cell Viability Assay | M | Colorimetric | 570 | - | V13154 |
| CyQUANT XTT Cell Viability Assay | M | Colorimetric | 450 | - | X12223 |
| *FC = flow cytometry; M = microplate assay **Ex = excitation; Em = emission | |||||
Metabolic activity reagents
Metabolic activity viability assays are live cell, membrane permeant reagents that detect cellular indicators such as the reducing environment or cellular redox potential. Metabolic activity assays comprise:
- alamarBlue/alamarBlue HS and PrestoBlue/PrestoBlue HS Cell Viability Reagents: These reagents are add-and-read, cell-permeable resazurin-based solutions that measure cell viability by using the reducing power of metabolically active cells. In live cells, resazurin is reduced to a red and highly fluorescent resorufin detectable on an absorbance- or fluorescence-based plate reader. alamarBlue HS and PrestoBlue HS have an improved purification process to remove various contaminants resulting in a >50% decrease in background fluorescence and >100% increase in signal-to-background ratio. Both assays are non-toxic and do not require cell lysis, as a result the reagent can be removed and replaced with complete growth media for further cell culturing.
Learn more: alamarBlue cell viability reagents
Learn more: PrestoBlue cell viability reagents
- CyQUANT MTT and XTT Cell Viability Assays: These viability assays are complete and optimized kits for the colorimetric detection of cell viability using the cellular redox potential in live cells. During this metabolic process, MTT is converted to an insoluble purple formazan product and XTT is converted to a water-soluble orange formazan product both of which are detectable on an absorbance-based microplate reader.
- Vybrant Cell Metabolic Assay Kit: This kit measures cell viability by reducing C12-resazurin to red-fluorescent C12-resorufin. C12-resazurin, a lipophilic version of resazurin, is more permeable to live cells and the reduced C12-resorufin product is better retained than the nonlipophilic version. This results in brighter signals and better detection limits when compared to resazurin alone.
Learn more: CYQUANT MTT and XTT cell viability assays
Learn more: Metabolic assays
Vybrant Cell Metabolic Assay Kit, with C12-resazurin
Vybrant Cell Metabolic Assay Kit determines cell viability through the reduction of non-fluorescent C12-resazurin to fluorescent C12-resorufin, with the signal of fluorescent resorufin being proportional to the number of live cells. Resazurin is non-toxic, allowing for the continuous monitoring of viability and can be multiplexed with other fluorescent dyes. Metabolic activity can be measure on a microplate reader or flow cytometer (Figure 10).
Figure 10. Vybrant cell metabolic assay kit. Flow cytometric analysis of Jurkat cells (T-cell leukemia, human) stained with C12-resazurin. Cells were incubated with 0.1 µM C12-resazurin, a component of the Vybrant Cell Metabolic Assay Kit, and 1 mM SYTOX Green for 15 minutes, then analyzed using 488 nm excitation. Healthy (live) cells reduce C12-resazurin to red-fluorescent C12-resorufin and exclude the cell-impermeant green-fluorescent SYTOX Green. Dead cells show little reduction of the C12-resazurin, but strong staining by SYTOX Green. Cells indicated in the figure as dying are of indeterminate viability, showing both reduction of C12-resazurin and compromised membrane integrity.
Ready-to-use viability dyes allow for easy and quick cell staining with no calculations, no dilutions, and no pipetting.
Learn more about these ready-to-use viability dyes
| Name | Platform* | Standard filter set | Laser (nm) | Ex/Em** (nm) | Cat. No. |
|---|---|---|---|---|---|
| NucGreen Dead 488 ReadyProbes Reagent | FC, FM | GFP | 488 | 504/523 | R37109 |
| NucRed Dead 647 ReadyProbes Reagent | FC, FM | Cy5 | 633 | 642/661 | R37113 |
| Propidium Iodide ReadyProbes | FC, FM | RFP | 488/532/561 | 535/617 | R37108 |
| NucRed Live 647 ReadyProbes | FC, FM | Cy5 | 633 | 638/686 | R37106 |
| NucBlue Live ReadyProbes (Hoechst 33342) | FC, FM | DAPI | UV | 360/460 | R37605 |
| SYTOX Green ReadyFlow | FC | - | 488 | 504/523 | R37168 |
| Propidium Iodide ReadyFlow | FC | - | 488/532/561 | 535/617 | R37169 |
| TO-PRO-3 ReadyFlow | FC | - | 633 | 642/661 | R37170 |
| SYTOX AADvanced ReadyFlow | FC | - | 488/532/561 | 546/647 | R37173 |
| *FC = flow cytometry; FM = fluorescence microscopy **Ex = excitation; Em = emission | |||||
Ready-to-use viability dyes
Ready-to-Use Viability Dyes provide an easy-to-use, liquid formulation in a convenient dropper bottle format for your everyday cell viability detection needs. Stable at room temperature, these reagents allow for storage at your bench, microscope, or flow cytometer for easy access. Rapid staining of cells with no washing required allows for convenient use throughout your experiment.
ReadyProbes reagents are available in blue and red fluorescence for use in fluorescence microscopy (Figure 11) and flow cytometry. These reagents are also available in multi-parameter kits (see Selection guide of cell viability assays).
Figure 11. NucRed Dead 647 ReadyProbes reagent. An 8 µm section of FFPE rat uterine tissue was deparaffinized and stained with NucRed Dead 647 ReadyProbes Reagent for 20 minutes then rinsed with PBS and mounted in ProLong Gold antifade. Imaged using a 20X objective and SemRock Far Red Briteline filter set.
Ready Flow reagents are exclusively used to determine cell viability in flow cytometry experiments (Figure 12). Compatible with the blue, green, yellow, and red laser lines, these dyes offer the flexibility for multiplex experiments.
Learn more: Ready-to-use flow cytometry reagents
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Figure 12. Viability analysis with SYTOX AADvanced Ready Flow Reagent. Jurkat cells, a human T cell leukemia cell line, were heat-killed and a 50:50 mixture of live/dead cells created. Cells were stained by adding 2 drops of SYTOX AADvanced Ready Flow Reagent to 1 x 106 cells (in 1 mL). The cells were then incubated for 15 minutes at 25°C. Data were acquired on an Attune NxT Flow Cytometer using a 488 nm laser. Emission was collected using a 695/40 nm filter.
Cell viability kits allow for the multi-parameter detection of cell viability.
Learn more about these cell viability kits.
| Name | Platform* | Ex/Em** (nm) | Cat. No. |
|---|---|---|---|
| ReadyProbes Cell Viability Imaging Kit (Blue/Green) | FC, FM, M | 360/460 504/523 | R37609 |
| ReadyProbes Cell Viability Imaging Kit (Blue/Red) | FC, FM, M | 360/460 535/617 | R37610 |
| Single-Channel Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 and SYTOX Green | FC | 499/521 503/524 | V13240 |
| Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 & Propidium Iodide (PI) | FC, FM | 499/521 535/617 | V13241 |
| Dead Cell Apoptosis Kit with Annexin V FITC and PI | FC | 494/518 535/617 | V13242 |
| Vybrant Apoptosis Assay Kit #4, YO-PRO-1/ Propidium Iodide | FC | 491/509 535/617 | V13243 |
| Vybrant Apoptosis Assay Kit #5, Hoechst 33342/ Propidium Iodide | FC | 350/461 535/617 | V13244 |
| Vybrant Apoptosis Assay Kit #6, Biotin-X Annexin V/ Alexa Fluor 350 Streptavidin/ Propidium Iodide | FC | 346/442 535/617 | V23200 |
| Chromatin Condensation & Membrane Permeability Dead Cell Apoptosis Kit with Hoechst 33342, YO-PRO-1, and PI | FC | 350/461 491/509 535/617 | V23201 |
| Dead Cell Apoptosis Kit with Annexin V PE and SYTOX Green | FC, FM | 503/524 488/575 | V35112 |
| Dead Cell Apoptosis Kit with Annexin V APC and SYTOX Green | FC, FM | 503/524 650/660 | V35113 |
| Metabolic Activity Dead Cell Apoptosis Kit with C12 Resazurin, Annexin V APC, and SYTOX Green | FC, FM | 503/524 571/585 650/660 | V35114 |
| Membrane Permeability Dead Cell Apoptosis Kit with PO-PRO-1 and 7-AAD | FC, FM | 434/456 546/647 | V35123 |
| Vybrant DyeCycle Violet/ STYOX AADvanced Apoptosis Kit | FC | 369/437 546/647 | A35135 |
| Pacific Blue Annexin V/ SYTOX AADvanced Apoptosis Kit | FC | 415/455 546/647 | A35136 |
| Violet Ratiometric Membrane Asymmetry Probe/ Dead Cell Apoptosis Kit | FC | 405/530 546/647 | A35137 |
| eBioscience Annexin V Apoptosis Detection Kit PE | FC, FM | 565/578 546/647 | 88-8102-72 |
| HCS Mitochondrial Health Kit | FM, M | 350/461 488/515 550/580 | H10295 |
| LIVE/DEAD cell viability kits | |||
| LIVE/DEAD Viability/Cytotoxicity Kit | FC, FM, M | 494/517 528/617 | L3224 |
| LIVE/DEAD Cell-Mediated Cytotoxicity Kit | FC, FM, M | 484/501 536/617 | L7010 |
| LIVE/DEAD Sperm Viability Kit | FC, FM | 485/517 586/617 | L7011 |
| LIVE/DEAD Reduced Biohazard Cell Viability Kit #1 | FC, FM, M | 488/505 535/624 | L7013 |
| LIVE/DEAD Cell Viability Assay Kit, C12 Resazurin/SYTOX Green | FC, FM, M | 504/523 563/587 | L34951 |
| The LIVE/DEAD Viability/Cytotoxicity Assay Kit (Green/Deep Red) | FM, M | 494/517 660/682 | L32250 |
| LIVE/DEAD Violet Viability/Vitality Kit | FC | 367/528 400/452 | L34958 |
| LIVE/DEAD Cell Imaging Kit | FM | 488/515 570/602 | R37601 |
| HCS LIVE/DEAD Green Kit | FM, M | 350/461 488/515 638/686 | H10290 |
| LIVE/DEAD bacterial viability kits | |||
| LIVE/DEAD BacLight Bacterial Viability Kit | FC, FM, M | 480/500 490/635 | L7007 |
| LIVE/DEAD BacLight Bacterial Viability Kit | FC, FM, M | 480/500 490/635 | L7012 |
| LIVE/DEAD BacLight Bacterial Viability Kit | FC, FM, M | 480/500 490/635 | L13152 |
| LIVE/DEAD BacLight Bacterial Viability and Counting Kit | FC | 485/498 535/617 | L34856 |
| FilmTracer LIVE/DEAD Biofilm Viability Kit | FC, FM, M | 480/500 490/635 | L10316 |
| LIVE/DEAD fungi and yeast viability kits | |||
| LIVE/DEAD Yeast Viability Kit | FM, M | 488/530 488/560-610 365/440 | L7009 |
| LIVE/DEAD FungaLight Yeast Viability Kit | FC | 485/498 535/617 | L34952 |
| *FC = flow cytometry; FM = fluorescence microscopy; M = microplate assay **Ex = excitation; Em = emission | |||
Cell viability kits
Multi-parameter assays contain multiple dyes within one convenient kit. Cell viability kits offer the convenience of determining viability by detecting membrane integrity in both live and dead cells, or through a combination of cellular function and membrane integrity. Each kit also provides the ability to be multiplexed with additional fluorescent dyes.
Cell viability assays using membrane integrity dyes to identify early apoptosis
7-AAD and PI can be used with Annexin V to distinguish early apoptotic cells from dead cells (Figure 13, 14). Early apoptosis is measured using fluorescently labeled Annexin V which detects phosphatidylserine expression on the cell membrane, while the addition of 7-AAD or PI identifies dead and necrotic cells.
Learn more: Annexin V staining
Learn more: Apoptosis assays
Figure 13. Identification of apoptotic cells using PI and Annexin V. Jurkat cells (T cell leukemia, human) treated with 10 μM camptothecin for 4 hours (right panel) or untreated (as control, left panel). Cells were then treated with Annexin V, Alexa Fluor 488 conjugate to identify apoptotic cells and with propidium iodide to identify dead cells, followed by flow cytometric analysis. Note that the camptothecin-treated cells (right panel) have a higher percentage of apoptotic cells (indicated by an “A”) than the basal level of apoptosis seen in the control cells (left panel). V = viable cells, D = dead cells.
Figure 14. Identification of apoptotic cells using Annexin V and 7-AAD. Mouse thymocytes were prepared as a single cell suspension and incubated overnight at 37°C in medium. Cells were harvested and stained with the Annexin V Apoptosis Detection Kit PE. Total cells were used for analysis.
Cell viability assays using membrane integrity dyes and mitochondrial membrane potential
HCS Mitochondrial Health Kit (Figure 14) measures two important cell-health parameters: mitotoxicity and cytotoxicity. Mitotoxicity is measured with the MitoHealth stain which detects changes in mitochondrial membrane potential, with loss of potential resulting in loss of signal. Cytotoxicity is measured with Image-It DEAD Green, a cell-impermeant dye that crosses the compromised plasma membrane of dead or dying cells and binds DNA to become highly fluorescent. Hoechst 3342 is a blue-fluorescent nuclear segmentation dye that binds DNA in live and dead cells.
Click image to enlarge
Figure 15. Multiplex analysis of cell loss, mitochondrial membrane potential, and plasma membrane permeability. HepG2 cells were plated on collagen-coated plates, treated with various doses of valinomycin for 24 hours, and stained with the Image-iT DEAD Green and MitoHealth stains (components of the HCS Mitochondrial Health Kit) for 30 minutes. The cells were then fixed and counterstained with Hoechst nuclear stain. Imaging and analysis were performed on the Thermo Scientific Cellomics ArrayScan VTI. (A) Images at selected concentrations of valinomycin. (B) The concentrations of valinomycin were used to calculate EC50 values for cell loss, mitochondrial membrane potential, and plasma membrane permeability.
LIVE/DEAD cell viability assays
LIVE/DEAD cell viability assays simultaneously detect live and dead populations based on membrane integrity, esterase activity, and/or structural segmentation (Figure 16). These fluorescence-based assays are available for use in cells, bacteria, yeast, and fungi. Specific assays can be used across one or multiple detection platforms including fluorescence microscopy, flow cytometry, or microplate reader. Each kit contains fluorescent dyes that can range from blue to near-IR emission allowing for multiplex capabilities.
Learn more: LIVE/DEAD cell viability assays
Figure 16. Tamoxifen-treated Hep G2 cells stained using the LIVE/DEAD cell imaging kit. Hep G2 cells grown in 96-well microplates were treated with 10 uM tamoxifen overnight, then stained with LIVE/DEAD Cell Imaging kit and imaged on a FLoid Cell Imaging Station. Bright field overlay on dead cells stained red and live cells stained green.
Nucleic acid binding dyes are cell-impermeant, nucleic acid-binding stains that are only able to enter cells with a compromised plasma membrane. They are dimly fluorescent in aqueous medium, but they highly fluoresce when bound to double-stranded DNA or RNA.
Learn more about these membrane integrity dyes
| Platform* | Standard filter set | Laser (nm) | Ex/Em** (nm) | Cat. No. | |
|---|---|---|---|---|---|
| Propidium iodide (PI) | FC, FM | RFP | 488/532/561 | 535/617 | P1304MP |
| TO-PRO-3 Iodide | FC, FM | Cy5 | 633 | 642/661 | T3605 |
| 7-AAD | FC, FM | Texas Red | 561 | 546/647 | A1310 |
| SYTOX Blue Nucleic Acid Stain | FC, FM | BFP | 405 | 444/480 | S11348 |
| SYTOX Green Nucleic Acid Stain | FC, FM | GFP | 488 | 483/503 | S7020 |
| SYTOX Orange Nucleic Acid Stain | FC, FM | RFP | 561 | 547/570 | S11368 |
| SYTOX Deep Red Nucleic Acid Stain | FC, FM, M | Cy5 | 633 | 660/682 | S11381 |
| SYTOX Blue Dead Cell Stain | FC, FM | - | 405 | 444/480 | S34857 |
| SYTOX Green Dead Cell Stain | FC, FM | - | 488 | 504/523 | S34860 |
| SYTOX Orange Dead Cell Stain | FC, FM | - | 561 | 547/570 | S34861 |
| SYTOX AADvanced Dead Cell Stain Kit | FC | - | 561 | 546/647 | S10349 |
| SYTOX Red Dead Cell Stain | FC, FM | - | 633 | 640/658 | S34859 |
| SYTOX Dead Cell Stain sampler pack | FC, FM | - | 488, 532, 561, 633 | multiple | S34862 |
| *FC = flow cytometry; FM = fluorescence microscopy; M = microplate assay **Ex = excitation; Em = emission | |||||
Membrane integrity viability dyes (nucleic acid binding)
Membrane integrity is a commonly measured parameter in cell viability assays. Membrane integrity dyes are cell-impermeant and only able to enter cells with a compromised plasma membrane. They are not able to penetrate the uncompromised plasma membrane of live cells. Membrane integrity viability dyes comprise:
- Nucleic acid-binding dyes (this section): These viability dyes are non-fluorescent in aqueous media but exhibit increased fluorescence upon binding to double-stranded DNA (dsDNA) or RNA. Nucleic-acid viability dyes include SYTOX dead cell stains, propidium iodide (PI), and 7-aminoactinomycin D (7-AAD).
- Amine-reactive dyes (next section: Membrane integrity viability dyes (amine-reactive)): These viability dyes covalently bind to the primary amine groups available on cellular proteins that are located on the exterior and interior of cells. When bound to surface amine proteins only, the resulting signal is dim; however, when the dye enters cells with a compromised plasma membrane and reacts with interior amine cellular proteins, the resulting signal is highly intense. Amine-reactive viability dyes include the LIVE/DEAD Fixable Dead Cell Stains for flow cytometry.
PI and 7-AAD
PI and 7-AAD are known as classic DNA-binding dyes commonly used in flow cytometry; however, both can also be used in fluorescence microscopy.
PI is a nuclear and chromosome stain commonly used to detect dead cells. In aqueous solution, PI has an excitation/emission spectra of 493/636; however, upon binding DNA, its fluorescence is enhanced 20- to 30-fold to an excitation/emission maximum of 535/617. This dye is commonly used in combination with apoptosis dyes such as annexin V; more information can be found in the Cell Viability Kits section below. With the addition of RNase, PI can also be used for DNA content cell cycle analysis in fixed cells (Figure 1).
Learn more: Cell cycle analysis assays
Figure 1. Detection of proliferation in Wil2S Lymphoma B cells. Cells were treated with 10 µM 5-bromo-2´-deoxyuridine (BrdU) in culture medium for one hour, then pelleted and fixed with cold 70% ethanol. After treatment with RNase and 4 M HCl (to denature the DNA), the cells were labeled with anti-BrdU and detected using green-fluorescent Alexa Fluor 488 Goat Anti–Mouse IgG antibody. In addition, the cells were labeled with red-fluorescent propidium iodide to assess the total cellular DNA content. The cells were analyzed by flow cytometry using 488 nm excitation; the fluorescent signals were collected at ~525 nm for the Alexa Fluor 488 dye and at ~675 nm for propidium iodide. Increased BrdU incorporation is indicative of actively proliferating cells.
7-AAD is another nuclear and chromatin dye that undergoes a spectral shift after binding to DNA (Figure 2). It selectively binds to GC regions of DNA resulting in a distinct banding pattern which allows for its use in chromosome banding studies. 7-AAD can also be used in cell cycle analysis for flow cytometry, and in cells that have fixed and permeabilized.
Figure 2. Wheat root tips in seven stages of the cell cycle. 7-aminoactinomycin D. Panel of confocal micrographs showing cells from wheat root tips in seven stages of the cell cycle. DNA was stained with 7-aminoactinomycin D, and microtubules were labeled with an anti-ß-tubulin antibody in conjunction with a fluorescein-labeled secondary antibody. Cells vary in width from about 15 µm to about 25 µm. The stages are (from top left): interphase cortical microtubule array; pre-prophase band of microtubules (predicts future plane of division); metaphase mitotic spindle; telophase, showing early phragmoplast and cell plate; fully developed phragmoplast during cytokinesis; late cytokinesis (plane of division matching plane of earlier pre-prophase band); restoration of cortical arrays in daughter cells. Image contributed by B.E.S. Gunning, Plant Cell Biology Group, Research School of Biological Sciences, Australian National University. Used with permission from Gunning, B.E.S. and Steer, M.W., Plant Cell Biology: Structure and Function, Jones and Bartlett Publishers (1995).
SYTOX Dead Cell Stains
SYTOX dead cell stains are easy-to-use, cell-impermeant dyes with increased fluorescence upon binding to dsDNA. These viability dyes can be used in cells without an additional wash step and visualized with minimal background staining since they are non-fluorescent in aqueous media. Available in multiple single-color formats and compatible with multiple lasers, these dyes allow the flexibility needed for multiplex experiments and across different platforms including fluorescence microscopy (Figure 3), flow cytometry (Figure 4), and microplates.
Learn more: Nucleus/nucleolus structure dyes
Click image to enlarge
Figure 3. Bovine pulmonary artery endothelial cells (BPAEC). MitoTracker Red CMXRos, SYTOX Green nucleic acid stain, biotin-XX goat anti–mouse IgG antibody and Cascade Blue NeutrAvidin biotin-binding protein. Bovine pulmonary artery endothelial cells (BPAEC) incubated with the fixable, mitochondrion-selective MitoTracker Red CMXRos. After staining, the cells were formaldehyde-fixed, acetone-permeabilized, treated with DNase-free RNase and counterstained using SYTOX Green nucleic acid stain. Microtubules were labeled with a mouse monoclonal anti–ß-tubulin antibody, biotin-XX goat anti–mouse IgG antibody and Cascade Blue NeutrAvidin biotin-binding protein. This photograph was taken using multiple exposures through bandpass optical filters appropriate for Texas Red dye, fluorescein and DAPI using a Nikon Labophot 2 microscope equipped with a Quadfluor epi-illumination system.
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Figure 4. Viable cell gating with SYTOX Dead Cell Stains. A mixture of heat-treated and untreated human peripheral blood leukocytes was stained with Invitrogen Alexa Fluor 488 dye–conjugated anti–human CD3 antibody, R-PE–conjugated anti–human CD8 antibody, and 5 nM SYTOX Red Dead Cell Stain before analysis by flow cytometry using 488 nm and 635 nm excitation. (A) Histogram showing distribution of two cell populations—dead cells that exhibit significant red fluorescence signal, and live cells, which do not. (B) Dual-parameter plot of CD8 and CD3 staining after gating on live cells.
Membrane integrity viability dyes (amine-reactive)
Amine-reactive viability dyes are cell-impermeant and react with cellular amine proteins. On live cells, these dyes bind to extracellular primary amines and dimly fluoresce; in dead cells, they bind to intracellular amine proteins resulting in a significant increase in cellular fluorescence. Since the binding to cellular proteins is covalent, the samples can be fixed and permeabilized without losing their viability staining pattern.
Image-iT DEAD Green
Image-iT DEAD Green (Figure 5) is another easy-to-use cell-impermeant, membrane integrity dye used to identify dead cells. This viability stain can be used in cells that will undergo fixation and/or permeabilization and allows for multiplex experiments with other fluorescent dyes.
Click image to enlarge
Figure 5. Single-channel viability assay with multiplexing. HeLa cells treated with camptothecin and stained with Hoechst 33342 (blue channel) for a total cell count and dead cells are stained with Image-iT DEAD Green (green channel).
Amine-reactive viability dyes are cell-impermeant and react with cellular amine proteins. Dimly fluorescent when bound to extracellular amine proteins on live cells, there is a significant increase in fluorescence when these dyes enter dead cells and bind to intracellular amine proteins. Since this binding is covalent, these samples can be fixed and permeabilized without losing their viability staining pattern.
Learn more about these fixable viability dyes
| Platform* | Standard filter set | Laser (nm) | Ex/Em** (nm) | Cat. No. | |
|---|---|---|---|---|---|
| Image-iT DEAD Green Viability Stain | FC, FM | GFP | 488 | 488/515 | I10291 |
| LIVE/DEAD Fixable Blue Stain | FC | - | UV | 350/450 | L34961 |
| LIVE/DEAD Fixable Violet Stain | FC | - | 405 | 416/451 | L34963 |
| LIVE/DEAD Fixable Lime Stain | FC | - | 405 | 405/506 | L34989 |
| LIVE/DEAD Fixable Aqua Stain | FC | - | 405 | 367/526 | L34965 |
| LIVE/DEAD Fixable Yellow Stain | FC | - | 405 | 400/575 | L34967 |
| LIVE/DEAD Fixable Green Stain | FC | - | 488 | 495/520 | L34969 |
| LIVE/DEAD Fixable Olive Stain | FC | - | 488 | 479/557 | L34977 |
| LIVE/DEAD Fixable Orange Stain | FC | - | 561 | 578/602 | L34983 |
| LIVE/DEAD Fixable Red Stain | FC | - | 488, 561 | 595/615 | L34971 |
| LIVE/DEAD Fixable Far Red Stain | FC | - | 633 | 650/665 | L34973 |
| LIVE/DEAD Fixable Scarlet Stain | FC | - | 633 | 702/723 | L34986 |
| LIVE/DEAD Fixable Near-IR (775) Stain | FC | - | 633 | 750/775 | L34975 |
| LIVE/DEAD Fixable Near-IR (780) Stain | FC | - | 633 | 633/785 | L34992 |
| LIVE/DEAD Fixable Near IR (876) Stain | FC | - | 808 | 840/876 | L34980 |
| *FC = flow cytometry; FM = fluorescence microscopy **Ex = excitation; Em = emission | |||||
LIVE/DEAD Fixable Dead Cell Stains
LIVE/DEAD Fixable Dead Cell Stains are cell-impermeant, amine-reactive dead cell stains optimal for use in cells that will undergo fixation and/or permeabilization. These stains are used exclusively for determining cell viability in flow cytometry (Figure 6). Available in multiple single-color formats and compatible with multiple lasers, these dyes provide the flexibility needed for multiplex experiments.
Learn more about Fixable Viability Dyes for Flow Cytometry
Click image to enlarge
Figure 6. Retention of LIVE/DEAD Fixable Viability Stains after fixation. The LIVE/DEAD Fixable Red (615) Viability kit for 488 and 561 nm excitation was used to differentially stain a mixture of live (left peak) and heat-treated Jurkat cells (right peak). Cells in (A) were not fixed; cells in (B) were fixed in 3.7% formaldehyde following staining. Samples were analyzed by flow cytometry using 488 nm excitation and ~615 nm emission.
Enzyme activity viability substrates are nonfluorescent reagents that react with cellular enzymes in live cells (CellTrace Calcein AM dyes) or react with cellular enzymes released from damaged cells (CyQUANT Cytotoxicity Assays) and use a fluorescent or colorimetric detection method.
Learn more about these enzyme activity substrates.
| Name | Platform* | Detection method | Absorbance (nm) | Fluorescence Ex/Em** (nm) | Cat. No. |
|---|---|---|---|---|---|
| CellTrace Calcein Blue, AM | FC, FM | Fluorescence | - | 323/439 | C34853 |
| CellTrace Calcein Violet, AM | FC, FM | Fluorescence | - | 400/452 | C34858 |
| CellTrace Calcein Green, AM | FC, FM | Fluorescence | - | 495/515 | C34852 |
| CellTrace Calcein Red-Orange, AM | FC, FM | Fluorescence | - | 577/590 | C34851 |
| Calcein, AM | FM | Fluorescence | - | 494/517 | C3100MP |
| CyQUANT LDH Cytotoxicity Assay - Fluorescence | M | Fluorescence | - | 560/590 | C20302 |
| CyQUANT Cytotoxicity Assay Kit (G6PD Release Assay) | M | Colorimetric, fluorescence | 563 | 560/590 | V23111 |
| CyQUANT LDH Cytotoxicity Assay | M | Colorimetric | 490 | - | C20300 |
| *FC = flow cytometry; FM = fluorescence microscopy; M = microplate assay **Ex = excitation; Em = emission | |||||
Enzymatic activity substrates
Enzymatic activity substrates are fluorogenic reagents that are non-fluorescent and either react with cellular enzymes in live cells or cellular enzymes released from damaged cells to become fluorescent or colorimetric. Enzymatic activity substrates comprise:
- CellTrace Calcein AM dyes: These viability dyes are cell-permeant, fluorogenic substrates that react with intracellular enzymes in live cells to become fluorescent. Within live cells, the AM group is cleaved by active esterases, resulting in fluorescence. This AM group cannot be cleaved by dead cells. Calcein AM dyes are ideal for short-term staining since the dyes can be transported out of the cell within a few hours.
- CyQUANT cytotoxicity assays: These viability assays detect extracellular glucose-6-phosphate dehydrogenase (G6PD) or lactate dehydrogenase (LDH) released from damaged cells. CyQUANT Cytotoxicity Assay Kit (G6PD Release Assay) and CyQUANT LDH Cytotoxicity Assay detect G6PD or LDH, respectively, by an enzymatic process that leads to the reduction of resazurin into red-fluorescent resorufin. CyQUANT LDH Cytotoxicity Assay is a colorimetric assay that detects LDH by an enzymatic process that leads to the formation of a red formazan product.
Learn more: CyQUANT cytotoxicity assays
CellTrace Calcein AM
CellTrace Calcein AM dyes are cell-permeant substrates that identify live cells. These substrates measure both enzymatic activity and membrane integrity. The enzymatic activity of live cells allows the AM group to be cleaved by esterases resulting in fluorescence, while the intact cell membrane of live cells is required for the intracellular retention of the fluorescent dye. Available with blue, violet, and green fluorescence, these dyes facilitate the flexibility you need for multiplex experiments and can be used with fluorescence or confocal microscopy (Figure 7) or in flow cytometry experiments (Figure 8).
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Figure 7. Primary Rat Cortex Neurons (A1084001) were treated with Calcein AM and with the FluxOR Red reagent. After stimulation of the potassium channels, cell viability was determined by imaging with a FITC/Alexa Fluor 488 filter setting to detect the green emission from Calcein AM (top left panel). The potassium ion flux was detected using FluxOR Red and a TRITC/Alexa Fluor 555 filter set (top right panel). The overlay of Calcein AM and FluxOR Red confirms cell viability and potassium ion flux (bottom panel).
Figure 8. Calcein AM viability dye. BALB/c thymocytes were stained with 12.5 nM Calcein AM for 30 minutes at room temperature (left). Thymocytes were kept on ice overnight (shaded histogram) or cultured overnight at 37°C without (purple) or with (blue) 1 µM dexamethasone. Thymocytes cultured overnight without dexamethasone were also stained with 7-AAD allowing further discrimination between live and dead cells (right). Total cells were used for analysis.
Cell viability assays using esterase substrates and amine-reactive dyes
The LIVE/DEAD Violet Viability/Vitality Kit provides a two-color fluorescence assay based on the measure of two essential cell health parameters: plasma membrane integrity to measure dead cells and intracellular esterase activity to measure live cells (Figure 9). CellTrace Calcein Violet AM and LIVE/DEAD Fixable Aqua fluorescent reactive dye are optimal for this application as both stains utilize the violet laser, allowing other laser lines to be used with other fluorescent dyes.
Figure 9. Staining of a mixture of heat-killed and untreated Jurkat cells. Jurkat cells were stained according to the protocol in the LIVE/DEAD Violet Viability/Vitality Kit. Cells were analyzed using a flow cytometer equipped with a 405 nm laser and a 450/50 nm bandpass filter for calcein violet–labeled live cells (L), and a 525/50 nm bandpass filter for the aqua dye–labeled dead cells (D).
Metabolic activity viability assays are live-cell, membrane permeant reagents that detect the cellular reducing environment (alamarBlue, PrestoBlue) or the cellular redox potential (CyQUANT MTT or CyQUANT XTT) and use a fluorescent or colorimetric detection method.
Learn more about these metabolic activity reagents.
| Name | Platform* | Detection method | Absorbance (nm) | Fluorescent Ex/Em** (nm) | Cat. No. |
|---|---|---|---|---|---|
| alamarBlue Cell Viability Reagent | M | Fluorescence | 570 | 560/590 | DAL1025 |
| alamarBlue HS Cell Viability Reagent | M | Fluorescence | 570 | 560/590 | A50100 |
| PrestoBlue Cell Viability Reagent | M | Fluorescence | 570 | 560/590 | A13261 |
| PrestoBlue HS Cell Viability Reagent | M | Fluorescence | 570 | 560/590 | P50200 |
| Vybrant Cell Metabolic Assay Kit, with C12-resazurin | FC, M | Fluorescence | 563 | 563/587 | V23110 |
| CyQUANT MTT Cell Viability Assay | M | Colorimetric | 570 | - | V13154 |
| CyQUANT XTT Cell Viability Assay | M | Colorimetric | 450 | - | X12223 |
| *FC = flow cytometry; M = microplate assay **Ex = excitation; Em = emission | |||||
Metabolic activity reagents
Metabolic activity viability assays are live cell, membrane permeant reagents that detect cellular indicators such as the reducing environment or cellular redox potential. Metabolic activity assays comprise:
- alamarBlue/alamarBlue HS and PrestoBlue/PrestoBlue HS Cell Viability Reagents: These reagents are add-and-read, cell-permeable resazurin-based solutions that measure cell viability by using the reducing power of metabolically active cells. In live cells, resazurin is reduced to a red and highly fluorescent resorufin detectable on an absorbance- or fluorescence-based plate reader. alamarBlue HS and PrestoBlue HS have an improved purification process to remove various contaminants resulting in a >50% decrease in background fluorescence and >100% increase in signal-to-background ratio. Both assays are non-toxic and do not require cell lysis, as a result the reagent can be removed and replaced with complete growth media for further cell culturing.
Learn more: alamarBlue cell viability reagents
Learn more: PrestoBlue cell viability reagents
- CyQUANT MTT and XTT Cell Viability Assays: These viability assays are complete and optimized kits for the colorimetric detection of cell viability using the cellular redox potential in live cells. During this metabolic process, MTT is converted to an insoluble purple formazan product and XTT is converted to a water-soluble orange formazan product both of which are detectable on an absorbance-based microplate reader.
- Vybrant Cell Metabolic Assay Kit: This kit measures cell viability by reducing C12-resazurin to red-fluorescent C12-resorufin. C12-resazurin, a lipophilic version of resazurin, is more permeable to live cells and the reduced C12-resorufin product is better retained than the nonlipophilic version. This results in brighter signals and better detection limits when compared to resazurin alone.
Learn more: CYQUANT MTT and XTT cell viability assays
Learn more: Metabolic assays
Vybrant Cell Metabolic Assay Kit, with C12-resazurin
Vybrant Cell Metabolic Assay Kit determines cell viability through the reduction of non-fluorescent C12-resazurin to fluorescent C12-resorufin, with the signal of fluorescent resorufin being proportional to the number of live cells. Resazurin is non-toxic, allowing for the continuous monitoring of viability and can be multiplexed with other fluorescent dyes. Metabolic activity can be measure on a microplate reader or flow cytometer (Figure 10).
Figure 10. Vybrant cell metabolic assay kit. Flow cytometric analysis of Jurkat cells (T-cell leukemia, human) stained with C12-resazurin. Cells were incubated with 0.1 µM C12-resazurin, a component of the Vybrant Cell Metabolic Assay Kit, and 1 mM SYTOX Green for 15 minutes, then analyzed using 488 nm excitation. Healthy (live) cells reduce C12-resazurin to red-fluorescent C12-resorufin and exclude the cell-impermeant green-fluorescent SYTOX Green. Dead cells show little reduction of the C12-resazurin, but strong staining by SYTOX Green. Cells indicated in the figure as dying are of indeterminate viability, showing both reduction of C12-resazurin and compromised membrane integrity.
Ready-to-use viability dyes allow for easy and quick cell staining with no calculations, no dilutions, and no pipetting.
Learn more about these ready-to-use viability dyes
| Name | Platform* | Standard filter set | Laser (nm) | Ex/Em** (nm) | Cat. No. |
|---|---|---|---|---|---|
| NucGreen Dead 488 ReadyProbes Reagent | FC, FM | GFP | 488 | 504/523 | R37109 |
| NucRed Dead 647 ReadyProbes Reagent | FC, FM | Cy5 | 633 | 642/661 | R37113 |
| Propidium Iodide ReadyProbes | FC, FM | RFP | 488/532/561 | 535/617 | R37108 |
| NucRed Live 647 ReadyProbes | FC, FM | Cy5 | 633 | 638/686 | R37106 |
| NucBlue Live ReadyProbes (Hoechst 33342) | FC, FM | DAPI | UV | 360/460 | R37605 |
| SYTOX Green ReadyFlow | FC | - | 488 | 504/523 | R37168 |
| Propidium Iodide ReadyFlow | FC | - | 488/532/561 | 535/617 | R37169 |
| TO-PRO-3 ReadyFlow | FC | - | 633 | 642/661 | R37170 |
| SYTOX AADvanced ReadyFlow | FC | - | 488/532/561 | 546/647 | R37173 |
| *FC = flow cytometry; FM = fluorescence microscopy **Ex = excitation; Em = emission | |||||
Ready-to-use viability dyes
Ready-to-Use Viability Dyes provide an easy-to-use, liquid formulation in a convenient dropper bottle format for your everyday cell viability detection needs. Stable at room temperature, these reagents allow for storage at your bench, microscope, or flow cytometer for easy access. Rapid staining of cells with no washing required allows for convenient use throughout your experiment.
ReadyProbes reagents are available in blue and red fluorescence for use in fluorescence microscopy (Figure 11) and flow cytometry. These reagents are also available in multi-parameter kits (see Selection guide of cell viability assays).
Figure 11. NucRed Dead 647 ReadyProbes reagent. An 8 µm section of FFPE rat uterine tissue was deparaffinized and stained with NucRed Dead 647 ReadyProbes Reagent for 20 minutes then rinsed with PBS and mounted in ProLong Gold antifade. Imaged using a 20X objective and SemRock Far Red Briteline filter set.
Ready Flow reagents are exclusively used to determine cell viability in flow cytometry experiments (Figure 12). Compatible with the blue, green, yellow, and red laser lines, these dyes offer the flexibility for multiplex experiments.
Learn more: Ready-to-use flow cytometry reagents
Click image to enlarge
Figure 12. Viability analysis with SYTOX AADvanced Ready Flow Reagent. Jurkat cells, a human T cell leukemia cell line, were heat-killed and a 50:50 mixture of live/dead cells created. Cells were stained by adding 2 drops of SYTOX AADvanced Ready Flow Reagent to 1 x 106 cells (in 1 mL). The cells were then incubated for 15 minutes at 25°C. Data were acquired on an Attune NxT Flow Cytometer using a 488 nm laser. Emission was collected using a 695/40 nm filter.
Cell viability kits allow for the multi-parameter detection of cell viability.
Learn more about these cell viability kits.
| Name | Platform* | Ex/Em** (nm) | Cat. No. |
|---|---|---|---|
| ReadyProbes Cell Viability Imaging Kit (Blue/Green) | FC, FM, M | 360/460 504/523 | R37609 |
| ReadyProbes Cell Viability Imaging Kit (Blue/Red) | FC, FM, M | 360/460 535/617 | R37610 |
| Single-Channel Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 and SYTOX Green | FC | 499/521 503/524 | V13240 |
| Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 & Propidium Iodide (PI) | FC, FM | 499/521 535/617 | V13241 |
| Dead Cell Apoptosis Kit with Annexin V FITC and PI | FC | 494/518 535/617 | V13242 |
| Vybrant Apoptosis Assay Kit #4, YO-PRO-1/ Propidium Iodide | FC | 491/509 535/617 | V13243 |
| Vybrant Apoptosis Assay Kit #5, Hoechst 33342/ Propidium Iodide | FC | 350/461 535/617 | V13244 |
| Vybrant Apoptosis Assay Kit #6, Biotin-X Annexin V/ Alexa Fluor 350 Streptavidin/ Propidium Iodide | FC | 346/442 535/617 | V23200 |
| Chromatin Condensation & Membrane Permeability Dead Cell Apoptosis Kit with Hoechst 33342, YO-PRO-1, and PI | FC | 350/461 491/509 535/617 | V23201 |
| Dead Cell Apoptosis Kit with Annexin V PE and SYTOX Green | FC, FM | 503/524 488/575 | V35112 |
| Dead Cell Apoptosis Kit with Annexin V APC and SYTOX Green | FC, FM | 503/524 650/660 | V35113 |
| Metabolic Activity Dead Cell Apoptosis Kit with C12 Resazurin, Annexin V APC, and SYTOX Green | FC, FM | 503/524 571/585 650/660 | V35114 |
| Membrane Permeability Dead Cell Apoptosis Kit with PO-PRO-1 and 7-AAD | FC, FM | 434/456 546/647 | V35123 |
| Vybrant DyeCycle Violet/ STYOX AADvanced Apoptosis Kit | FC | 369/437 546/647 | A35135 |
| Pacific Blue Annexin V/ SYTOX AADvanced Apoptosis Kit | FC | 415/455 546/647 | A35136 |
| Violet Ratiometric Membrane Asymmetry Probe/ Dead Cell Apoptosis Kit | FC | 405/530 546/647 | A35137 |
| eBioscience Annexin V Apoptosis Detection Kit PE | FC, FM | 565/578 546/647 | 88-8102-72 |
| HCS Mitochondrial Health Kit | FM, M | 350/461 488/515 550/580 | H10295 |
| LIVE/DEAD cell viability kits | |||
| LIVE/DEAD Viability/Cytotoxicity Kit | FC, FM, M | 494/517 528/617 | L3224 |
| LIVE/DEAD Cell-Mediated Cytotoxicity Kit | FC, FM, M | 484/501 536/617 | L7010 |
| LIVE/DEAD Sperm Viability Kit | FC, FM | 485/517 586/617 | L7011 |
| LIVE/DEAD Reduced Biohazard Cell Viability Kit #1 | FC, FM, M | 488/505 535/624 | L7013 |
| LIVE/DEAD Cell Viability Assay Kit, C12 Resazurin/SYTOX Green | FC, FM, M | 504/523 563/587 | L34951 |
| The LIVE/DEAD Viability/Cytotoxicity Assay Kit (Green/Deep Red) | FM, M | 494/517 660/682 | L32250 |
| LIVE/DEAD Violet Viability/Vitality Kit | FC | 367/528 400/452 | L34958 |
| LIVE/DEAD Cell Imaging Kit | FM | 488/515 570/602 | R37601 |
| HCS LIVE/DEAD Green Kit | FM, M | 350/461 488/515 638/686 | H10290 |
| LIVE/DEAD bacterial viability kits | |||
| LIVE/DEAD BacLight Bacterial Viability Kit | FC, FM, M | 480/500 490/635 | L7007 |
| LIVE/DEAD BacLight Bacterial Viability Kit | FC, FM, M | 480/500 490/635 | L7012 |
| LIVE/DEAD BacLight Bacterial Viability Kit | FC, FM, M | 480/500 490/635 | L13152 |
| LIVE/DEAD BacLight Bacterial Viability and Counting Kit | FC | 485/498 535/617 | L34856 |
| FilmTracer LIVE/DEAD Biofilm Viability Kit | FC, FM, M | 480/500 490/635 | L10316 |
| LIVE/DEAD fungi and yeast viability kits | |||
| LIVE/DEAD Yeast Viability Kit | FM, M | 488/530 488/560-610 365/440 | L7009 |
| LIVE/DEAD FungaLight Yeast Viability Kit | FC | 485/498 535/617 | L34952 |
| *FC = flow cytometry; FM = fluorescence microscopy; M = microplate assay **Ex = excitation; Em = emission | |||
Cell viability kits
Multi-parameter assays contain multiple dyes within one convenient kit. Cell viability kits offer the convenience of determining viability by detecting membrane integrity in both live and dead cells, or through a combination of cellular function and membrane integrity. Each kit also provides the ability to be multiplexed with additional fluorescent dyes.
Cell viability assays using membrane integrity dyes to identify early apoptosis
7-AAD and PI can be used with Annexin V to distinguish early apoptotic cells from dead cells (Figure 13, 14). Early apoptosis is measured using fluorescently labeled Annexin V which detects phosphatidylserine expression on the cell membrane, while the addition of 7-AAD or PI identifies dead and necrotic cells.
Learn more: Annexin V staining
Learn more: Apoptosis assays
Figure 13. Identification of apoptotic cells using PI and Annexin V. Jurkat cells (T cell leukemia, human) treated with 10 μM camptothecin for 4 hours (right panel) or untreated (as control, left panel). Cells were then treated with Annexin V, Alexa Fluor 488 conjugate to identify apoptotic cells and with propidium iodide to identify dead cells, followed by flow cytometric analysis. Note that the camptothecin-treated cells (right panel) have a higher percentage of apoptotic cells (indicated by an “A”) than the basal level of apoptosis seen in the control cells (left panel). V = viable cells, D = dead cells.
Figure 14. Identification of apoptotic cells using Annexin V and 7-AAD. Mouse thymocytes were prepared as a single cell suspension and incubated overnight at 37°C in medium. Cells were harvested and stained with the Annexin V Apoptosis Detection Kit PE. Total cells were used for analysis.
Cell viability assays using membrane integrity dyes and mitochondrial membrane potential
HCS Mitochondrial Health Kit (Figure 14) measures two important cell-health parameters: mitotoxicity and cytotoxicity. Mitotoxicity is measured with the MitoHealth stain which detects changes in mitochondrial membrane potential, with loss of potential resulting in loss of signal. Cytotoxicity is measured with Image-It DEAD Green, a cell-impermeant dye that crosses the compromised plasma membrane of dead or dying cells and binds DNA to become highly fluorescent. Hoechst 3342 is a blue-fluorescent nuclear segmentation dye that binds DNA in live and dead cells.
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Figure 15. Multiplex analysis of cell loss, mitochondrial membrane potential, and plasma membrane permeability. HepG2 cells were plated on collagen-coated plates, treated with various doses of valinomycin for 24 hours, and stained with the Image-iT DEAD Green and MitoHealth stains (components of the HCS Mitochondrial Health Kit) for 30 minutes. The cells were then fixed and counterstained with Hoechst nuclear stain. Imaging and analysis were performed on the Thermo Scientific Cellomics ArrayScan VTI. (A) Images at selected concentrations of valinomycin. (B) The concentrations of valinomycin were used to calculate EC50 values for cell loss, mitochondrial membrane potential, and plasma membrane permeability.
LIVE/DEAD cell viability assays
LIVE/DEAD cell viability assays simultaneously detect live and dead populations based on membrane integrity, esterase activity, and/or structural segmentation (Figure 16). These fluorescence-based assays are available for use in cells, bacteria, yeast, and fungi. Specific assays can be used across one or multiple detection platforms including fluorescence microscopy, flow cytometry, or microplate reader. Each kit contains fluorescent dyes that can range from blue to near-IR emission allowing for multiplex capabilities.
Learn more: LIVE/DEAD cell viability assays
Figure 16. Tamoxifen-treated Hep G2 cells stained using the LIVE/DEAD cell imaging kit. Hep G2 cells grown in 96-well microplates were treated with 10 uM tamoxifen overnight, then stained with LIVE/DEAD Cell Imaging kit and imaged on a FLoid Cell Imaging Station. Bright field overlay on dead cells stained red and live cells stained green.
Tools and resources
SpectraViewer
Supports all levels of experimental complexity, use the tool to compare excitation and emission spectra of fluorophores and reagents.
Flow cytometry protocols handbook
Protocols that fit your needs in flow cytometry ranging from sample preparation to numerous cell stimulation conditions, staining, immunophenotyping, and data analysis strategies.
Imaging protocol handbook
Protocols that fit your needs in imaging ranging from sample and assay preparation to staining, labeling, and data analysis strategies.
Citations
- Kamiloglu S; Sari G; Ozdal T; Capanoglu E (2020) Food Frontiers 1:332-349.
- Alamoudi WA, Ahmad F, Acharya S, et al (2108) J BUON 23(5):1505-1513. PMID: 30570879.
- Riss TL, Moravec RA, Niles AL, et al. Cell Viability Assays. (2013) [Updated 2016]. In: Markossian S, Sittampalam GS, Grossman A, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-. PMID: 23805433.
Resources
Flow Cytometry Panel Builder—Design your flow cytometry panel with this online tool for a simplified, customizable experience to fit your needs
Cell Viability and Function Information – Learn about assays and techniques for evaluating cell health and function using various detection methods.
Cell Analysis Learning Center – Access educational resources to learn about applications, techniques, and basic principles during experiment planning and execution.
Related pages to cell viability
Support
Cell Analysis Support Center—Find technical information, tips and tricks, and answers to everyday problems
5 Steps resources
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