Measuring Plasma Membrane Integrity
Plasma membrane integrity is the most widely measured parameter for determining cell viability. These methods rely on the principle that viable cells have intact plasma membranes and are impermeable to polar dyes, whereas dead cells are not. The compromised membrane integrity of dead cells allows these dyes to penetrate the membrane and stain intracellular components, such as nucleic acids and proteins. Stained dead cells can then be identified and removed from the final analysis by gating on the unstained, live cell population. Membrane integrity dyes are simple-to-use, reliable, and can be combined with intracellular esterase substrates, membrane potential-sensitive dyes, and organelle probes for multiplex analysis. Common membrane integrity dyes include trypan blue vital stains, nucleic acid binding dyes, and amine-reactive viability dyes.
Trypan Blue Dye Exclusion Assays
Illustration of trypan blue dye exclusion assay workflow (created in BioRender).
One of the earliest and most common methods for measuring cell viability is the trypan blue dye exclusion assay. Trypan blue
is a membrane-impermeant stain that selectively penetrates dead cells and binds to intracellular proteins, rendering their cytoplasm blue. Cell suspensions assayed with trypan blue ideally display two readouts, live-cell populations with clear cytoplasms and dead cell populations with blue cytoplasms, that can be easily identified and counted via optical microscopy. While trypan blue dye exclusion assays are inexpensive and simple to perform, trypan blue is recognized as a carcinogen and must be handled with care and disposed of properly. Trypan UltraBlue™
, Trypan Purple™
, and Trypan Red Plus™
are less toxic alternatives to trypan blue and analogous in utility. Trypan UltraBlue™, Trypan Purple™, and Trypan Red Plus™ are highly purified and can be used up to 0.5 mM, 0.75 mM, or 1 mM, respectively, with minimal cell cytotoxicity.
Cells labeled with trypan vital stains should be loaded into a hemocytometer and examined immediately under a light microscope at low magnification, as longer incubations times will lead to increases in cell death and reduced viability counts. To calculate the percentage of viable cells, divide the total number of viable cells by the total number of cells and multiply by 100.
Table 1. Specifications for trypan blue vital stain and derivatives.
|Trypan Blue, sodium salt *10 mM aqueous solution*||∼960||607||10 mL||2453|
|Trypan Blue, sodium salt *UltraPure grade, Purified to eliminate fluorescent impurities*||∼960||607||10 g||2452|
|Trypan Blue, sodium salt *CAS 72-57-1*||∼960||607||100 g||2450|
|Trypan UtraBlue™, sodium salt *0.1 M aqueous solution*||∼600||618||1 mL||2455|
|Trypan Purple™, *0.1 M aqueous solution*||∼600||586||10 mL||2465|
|Trypan Purple™, *0.1 M aqueous solution*||∼1000||586||100 mL||2466|
|Trypan Red Plus™, *0.1 M aqueous solution*||∼600||534||10 mL||2456|
|Trypan Red Plus™, *0.1 M aqueous solution*||∼600||534||100 mL||2457|
Nucleic Acid Viability Dyes
Nucleic acid viability dyes, such as propidium iodide (PI), 7-AAD, and Nuclear DCS1™ dead cell stains, are membrane-impermeant nucleic acid binding dyes for detecting dead cell populations by flow cytometry, fluorescence microscopy, and fluorescence-based microplate assays. These dyes, while excluded from viable cells, readily penetrate the compromised membranes of dead cells and selectively bind to DNA, whereby fluorescence and quantum yield dramatically increase. More importantly, because nucleic acid binding dyes are essentially non-fluorescent in the absence of DNA, no wash steps are required, and background interference is minimal. Nucleic acid binding dyes are frequently used with cell-permeable intracellular esterase substrates, membrane potential-sensitive probes (Mitochondrial Membrane Potential Probes
), and fluorescent ion indicators (Calcium Indicators
) to simultaneously detect live and dead cell populations.
Propidium Iodide, 7-Aminoactinomycin D and Cyanine Dyes for Measuring Dead Cells
PI and 7-AAD
Two-color discrimination of live and dead Bacillus subtilis
populations using the MycoLight™ Fluorescence Live/Dead Bacterial Imaging Kit (Cat No. 22411
). Live bacteria with active intracellular esterase were labeled with MycoLight™ 520 (green), while 70% alcohol-killed dead bacteria with compromised membranes were labeled with propidium iodide (red).
Cell-impermeable propidium iodide (PI)
and 7-aminoactinomycin D (7-AAD)
are red-fluorescent, intercalating dyes often used to detect dead cell populations by flow cytometry and fluorescence microscopy. PI binds to DNA and RNA by intercalating between the bases with little or no sequence preference, whereas 7-AAD intercalates in double-stranded DNA with a high affinity for GC-rich regions. Nevertheless, both undergo significant fluorescence enhancements upon binding DNA, with PI enhancing 20- to 30-fold. PI can be excited by a xenon-arc lamp, mercury-arc lamp, or by the 488 nm or 514 nm spectral lines of the argon-ion laser. Similarly, 7-AAD can be well-excited by the argon-ion laser. However, optimum excitation is achieved when using the 543 nm spectral line of the helium-neon laser. These stains exhibit significantly large Stokes shifts with PI emitting maximally at 618 nm and 7-AAD at 648 nm, making them useful for multicolor analysis with blue and green fluorescent probes.
Neither dye is fixable with glutaraldehyde or paraformaldehyde (PFA). However, they can be used in fixed and permeabilized cells for cell cycle analysis by DNA content measurement (i.e., PI will require the addition of RNase). Alternatively, the high affinity of 7-AAD for GC-rich regions of DNA makes it useful for chromosome banding studies. The binding of 7-AAD to DNA yields a distinct pattern in polytene chromosomes and chromatin.
Dimeric Cyanine Dyes
The dimeric cyanine dye series of DiTO™ and DiYO™ also display exceptional sensitivity for various nucleic acid species, including double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and RNA. They exhibit a 100 to 1000-fold fluorescence enhancement upon nucleic acid binding, and their molar extinction coefficients are an order magnitude greater than ethidium homodimers. DiYO™-1
dyes are optimally excited by the 488 nm and 514 nm spectral line of the argon-ion laser, respectively, and emits green fluorescence. DiYO™-3
dyes are well-excited by the He-Ne laser and emit red fluorescence. All of the dyes in the DiYO™ and DiTO™ series are supplied as 5 mM solutions in DMSO, except for DiYO™-1, which is also available as a solid. Cyanine monomer dyes TWO-PRO™ 1
and TWO-PRO™ 3
may also be used to analyze DNA content in dead cells.
Table 2. Specifications for PI, 7-AAD, and cyanine dimer dyes.
|Propidium Iodide||537||618||6,000||0.2||25 mg||17515|
|Propidium Iodide *10 mM aqueous solution*||537||618||6,000||0.2||1 mL||17517|
|7-AAD [7-Aminoactinomycin D]||549||648||27,500||0.02||1 mg||17501|
|DiTO™-1 [equivalent to TOTO®-1] *5 mM DMSO solution*||514||531||117,000||0.34||0.2 mL||17575|
|DiTO™-3 [equivalent to TOTO®-3] *5 mM DMSO solution*||642||661||154,000||0.06||0.2 mL||17576|
|DiYO™-1 [equivalent to YOYO®-1]||491||508||98,900||0.52||1 mg||17579|
|DiYO™-1 [equivalent to YOYO®-1] *5 mM DMSO solution*||491||508||98,900||0.52||0.2 mL||17580|
|DiYO™-3 [equivalent to YOYO®-3] *5 mM DMSO solution*||612||631||167,000||0.15||0.2 mL||17581|
|TWO-PRO™ 1 [equivalent to TO-PRO®-1] *5 mM DMSO solution*||515||531||62,800||0.25||0.2 mL||17571|
Nuclear DCS1™ Dead Cell Stains
Nuclear DCS1™ dead cell stains are a family of membrane-impermeant, high-affinity nucleic acid dyes for analyzing DNA content in dead, fixed, or permeabilized cells. These dyes easily penetrate the compromised membranes of dead cells and selectively bind to DNA, producing exceptionally bright signals. In aqueous media, Nuclear DCS1™ dyes are essentially non-fluorescent and can be conveniently added to cells without an additional wash step and imaged with minimal interference. The fluorescence absorption and emission spectra of Nuclear DCS1™ dyes span the visible-light spectrum from violet to red, making them compatible with various fluorescence platforms, including fluorescence microscopes, flow cytometers, and microplate readers. Moreover, the multiple single-color formats of these dyes provide the necessary flexibility to facilitate multicolor applications in imaging and flow cytometry. All dyes in the Nuclear DCS1™ series are supplied as 5 mM solutions in DMSO.
Nuclear Blue™ DCS1 nuclear counterstain (blue) with EpCAM Rabbit mAb and iFluor™ 555 goat anti-rabbit IgG (red). Formaldehyde fixed HeLa cells stained with Nuclear Green™ DCS1 (green) and Phalloidin-iFluor™ 680 conjugate (red). Formaldehyde fixed HeLa cells stained with Nuclear Red™ DCS1 (red) and Phalloidin-iFluor™ 488 conjugate (green).
Table 3. Specifications for Nuclear DCS1™ dead cell stains.
|Nuclear Violet™ DCS1||371||454||0.5 mL||17549|
|Nuclear Blue™ DCS1||348||469||0.5 mL||17548|
|Nuclear Green™ DCS1||503||527||0.5 mL||17550|
|Nuclear Orange™ DCS1||514||556||0.5 mL||17551|
|Nuclear Red™ DCS1||631||651||0.5 mL||17552|
Amine-Reactive Cell Meter™ Fixable Viability Dyes
Detection of Jurkat cell viability using Cell Meter™ BX590 fixable viability dye. Jurkat cells were treated and stained with Cell Meter™ BX590 (Cat No. 22514
), fixed in 3.7% formaldehyde, and analyzed by flow cytometry. The dead cell population (blue peak) is easily distinguished from the live cell population (red peak) with the PE-Texas Red channel. Nearly identical results were obtained before and after fixation.
Cell Meter™ fixable viability dyes are membrane-impermeant, amine-reactive dyes designed to discriminate live and dead cell populations in flow cytometry. These dyes readily penetrate dead cells with compromised membrane integrity and bind irreversibly to cellular amine proteins and other intracellular components producing significant cellular fluorescence. On live cells, these dyes are unable to penetrate the membrane leaving only cell-surface amines to bind with and dimly fluoresce. The difference in intensity between viable and dead cells is significant, making it easy to exclude dead cells from analysis. Because staining is robust and stable, cells labeled with Cell Meter™ fixable viability dyes can be fixed, permeabilized, and probed for intracellular immunophenotyping. The Cell Meter™ fixable viability dyes are available in 9 fluorescent stains to provide users greater flexibility in multicolor panel design. Dyes included in this series are excited by major excitation sources, including the 405, 488, 633, and 647 nm laser lines, and emit in common channels.
Table 4. Fixable viablity dyes for live/dead cell analysis during flow cytometry
|Cell Meter™ VX450 fixable viability dye||406||445||200 Tests||22540|
|Cell Meter™ VX500 fixable viability dye||433||498||200 Tests||22542|
|Cell Meter™ BX520 fixable viability dye||491||516||200 Tests||22510|
|Cell Meter™ BX590 fixable viability dye||492||579||200 Tests||22514|
|Cell Meter™ BX650 fixable viability dye||518||654||200 Tests||22520|
|Cell Meter™ RX660 fixable viability dye||649||664||200 Tests||22530|
|Cell Meter™ RX700 fixable viability dye||690||713||200 Tests||22532|
|Cell Meter™ RX780 fixable viability dye||629||767||200 Tests||22536|
|Cell Meter™ IX830 fixable viability dye||811||822||200 Tests||22529|
Measuring Enzymatic Activity in Live Cells
Live cells contain several non-specific cytosolic esterases that are responsible for catalyzing ester hydrolysis, a key reaction in cell and drug metabolism. The activity of these enzymes can be detected with a wide variety of fluorogenic esterase substrates, including calcein AM, BCECF AM, and carboxyfluorescein diacetate (CFDA), and used to identify live cell populations. As near-neutral compounds, esterase substrates diffuse freely and noninvasively across the plasma membrane and permeate the cytosol of live cells. Once inside the cell, cytosolic esterases rapidly hydrolyze these non-fluorescent substrates into highly fluorescent polar products that are retained by cells with intact membrane integrity. Labeled cells can be measured by fluorescence microscopy, flow cytometry, or in a fluorescence microplate reader, and can be combined with apoptotic and necrotic indicators for multiparameter analysis of cell health.
Calcein AM Dyes for Labeling Live Cells
Detection of Jurkat cell viability using calcein Blue AM. Jurkat cells were treated with staurosporine and stained with Calcein Blue AM (Cat No. 22007
, blue), annexin V iFluor™ 488 (Cat No. 20071
, green), and 7-AAD (Cat No. 17501
Calcein AM (i.e., a calcein dye synthesized with acetoxymethyl esters (AM)) is a cell-permeable, fluorogenic esterase substrate used to identify live cells. By modifying calcein and other polar compounds with AM groups, it promotes cell permeability by increasing their hydrophobicity and, more importantly, makes esterase hydrolyzation a requirement to activate their fluorescence. Following brief incubation and hydrolysis of calcein AM, viable cells will fluoresce bright green when excited by the 488 nm spectral line of the argon-ion laser or with any other blue light excitation source. Compared to other esterase substrates, such as BCECF AM or CFDA, the brighter fluorescence, photostability, and low cytotoxicity of calcein AM are advantageous for a variety of studies, including cell tracking, cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis, and cytotoxicity.
Consequently, the fluorescence emission maximum of calcein at ~521 nm makes it challenging to use in combination with GFP-transfected cells or other green fluorescent indicators for multiplex analysis. To address the single color limitation of calcein AM, AAT Bioquest has developed calcein derivatives in a broad range of color options, including Calcein Blue AM, Calcein Orange™ AM, Calcein Red™ AM, and Calcein Deep Red™ AM dyes for multicolor labeling and functional analysis of live cells. For instance, calcein Blue AM is a UV-light excitable viability indicator that emits fluorescence maximally at ~441 nm and is designed for use with instruments optimized for detecting blue fluorescence. It can be used in combination with the phosphatidylserine stain Annexin V iFluor™ 488 (Cat No. 20071) and nucleic acid dead cell stain 7-AAD for the three-color discrimination of live/apoptotic/necrotic cell populations.
CytoCalcein™ Violet Viability Dyes Optimized for Flow Cytometry
CytoCalcein™ Violet 450 and CytoCalcein™ Violet 500 are optimized for use in flow cytometry. The enzymatic conversion of non-fluorescent CytoCalcein™ Violet 450 AM and CytoCalcein™ Violet 500 AM to highly fluorescent CytoCalcein™ Violet 450 and CytoCalcein™ Violet 500 products (Ex/Em maxima ~406/445 nm and ~420/505 nm, respectively) are efficiently excited by the standard 405 nm violet laser found in most flow cytometers. In conjunction with red fluorescent nucleic acid dead cell stains propidium iodide (PI, Ex/Em maxima ~537/618 nm) or 7-Aminoactinomycin D (7-AAD, Ex/Em ~549/648 nm), CytoCalcein™ Violet dyes are excellent fluorophores for two-color discrimination of live from dead cell populations based on membrane integrity and esterase activity. Removing data points representing dead cells is a critical step for improving the accuracy of your flow cytometry analysis. Often dead cells will bind non-specifically to many reagents contributing to false-positive results.
Table 5. Specifications for calcein AM and calcein AM derivatives.
|Calcein Blue, AM *CAS 168482-84-6*||354 nm||441 nm||1 mg||22007|
|Calcein UltraBlue™||359 nm||458 nm||1 mg||21908|
|CytoCalcein™ Violet 450 *Excited at 405 nm*||406 nm||445 nm||1 mg||22012|
|CytoCalcein™ Violet 500 *Excited at 405 nm*||420 nm||505 nm||1 mg||22013|
|Calcein, AM *CAS 148504-34-1*||501 nm||521 nm||1 mg||22002|
|Calcein, AM *UltraPure grade* *CAS 148504-34-1*||501 nm||521 nm||1 mg||22003|
|Calcein, AM *UltraPure grade* *CAS 148504-34-1*||501 nm||521 nm||20x50 µg||22004|
|Calcein Orange™ diacetate||531 nm||545 nm||1 mg||22009|
|Calcein Red™ AM||562 nm||576 nm||1 mg||21900|
|Calcein Deep Red™ AM ester||643 nm||663 nm||1 mg||22011|
Metabolic indicators, such as tetrazolium salts and resazurin, measure the cellular reduction potential in metabolically active cells to evaluate viability, cytotoxicity, and proliferation. Following reduction, these substrates yield bright colorimetric products that can be measured using a spectrophotometer or microplate reader in low- or high-throughput configurations. The reduced form of resazurin can also emit fluorescence and be measured using a fluorescence microplate reader.
Tetrazolium Salts for Cell Viability
Cell proliferation was determined using Cell Meter™ Colorimetric MTT Cell Proliferation Kit. HeLa cells at 0 to 40,000 cells/well/100 µL were added to a clear bottom 96-well plate overnight. The absorbance was measured at 560 nm using a SpectraMax reader (Molecular Devices). 500 cells/well was detected compared to ~5,000 cells/well with Sigma's MTT assay kit.
The four types of tetrazolium salts used to measure viability are MTT, MTS, XTT, and WST-8. Of the four, MTT is the only one that is positively charged and can readily penetrate viable cells. The others (MTS, XTT, and WST-8), which are negatively charged and membrane-impermeant, require an intermediate electron acceptor that can transfer electrons from the cytoplasm or plasma membrane to facilitate reduction into colored formazan products. Reduction of MTT is the most common, however, for most commercial MTT assays, solubilization of the purple formazan product in DMSO or isopropanol is required before quantification. The convenient Cell Meter™ Colorimetric MTT Cell Proliferation Kit (Cat No. 22768) simplifies the task of counting cells to an easy mix and read format without the need for solubilization. The concentration of formazan determined by optical density at 560 nm is directly proportional to the number of live cells.
Cell proliferation was determined using Cell Meter™ Colorimetric WST-8 Cell Quantification Kit. HeLa cells at 0 to 10,000 cells/well/100 µL were added in a clear-bottom 96-well plate. The absorbance was measured at 460 nm using a SpectraMax reader (Molecular Devices).
Among the other tetrazolium salts, the water-soluble WST-8 offers the highest sensitivity. Reduction of WST-8 by cellular dehydrogenases yields water-soluble orange formazan crystals that do not require solubilization prior to quantification. The concentration of formazan product can be determined by measuring absorbance at 460 nm and is proportional to the number of living cells. The Cell Meter™ Colorimetric WST-8 Cell Quantification Kit provides a sensitive assay with exceptional linearity of up to ~106 cells per well. No pre-mixing of components is required, as the WST-8 substrate is conveniently provided in a solution that can be directly added to the cells. Each kit provides sufficient reagents for either ~1000 (22770) or ~5000 tests (22771) using a standard 96-well microplate. WST-8 is also available as a stand-alone reagent in 25 mg (Cat No. 15705), 100 mg (Cat No. 15706), and 1 g (Cat No. 15707) unit sizes supplied as 50 mM aqueous solutions.
Table 6. Colorimetric MTT and WST-8 tetrazolium salt-based assays for measuring cell proliferation.
|Cell Meter™ Colorimetric MTT Cell Proliferation Kit||1000 Tests||22768|
|Cell Meter™ Colorimetric MTT Cell Proliferation Kit||5000 Tests||22769|
|Cell Meter™ Colorimetric WST-8 Cell Quantification Kit||1000 Tests||22770|
|Cell Meter™ Colorimetric WST-8 Cell Quantification Kit||5000 Tests||22771|
|ReadiUse™ WST-8 *50 mM aqueous solution*||25 mg||15705|
|ReadiUse™ WST-8 *50 mM aqueous solution*||100 mg||15706|
|ReadiUse™ WST-8 *50 mM aqueous solution*||1 g||15707|
Resazurin, also known as Alamar Blue, is a water-soluble redox-sensitive indicator that can be used to measure the cell viability of mammalian cells, bacteria, and fungi. In live metabolically active cells, the blue-colored and non-fluorescent resazurin is irreversibly reduced to a pink-colored and highly-fluorescent resorufin by aerobic respiration. The signal output may be measured using a standard spectrophotometer at 570 nm or by fluorimetry using a 544/590 nm filter set and is proportional to the number of viable cells over a wide concentration range. Since resazurin is minimally toxic to live cells, it is helpful for quantitatively measuring cell proliferation and other long-term studies.
Table 7. Product specifications for resazurin.
|Resazurin, sodium salt *CAS 62758-13-8*||605||571/584||100 mg||15700|