section of flow cytometry histogram showing two distinct fluorescence peaks  

Dead cells often give false positive results, as they tend to bind nonspecifically to many reagents. Therefore, removing dead cells from your flow cytometry data is a critical step to help ensure accurate results and analysis. Molecular Probes® nonfixable cell viability assays for flow cytometry offer researchers options for distinguishing live and dead cell populations that are more accurate than forward- and side-scatter data.

Viability assays using cell-impermeant SYTOX® DNA-binding dyes

SYTOX® Dead Cell Stains do not cross intact cell membranes, and they exhibit increased fluorescence upon dsDNA binding, making them some of our most brilliant dead cell stains (Figure 1). Easy to use, SYTOX® Dead Cell Stains can be applied to cells and visualized without an additional wash step because they are nonfluorescent in aqueous media. Available in multiple single-color formats and compatible with multiple lasers, these dyes provide the flexibility you need. These stains can be used on multiple platforms, including fluorescence microscopy, flow cytometry, and microplates.

Selection guide for cell-impermeant SYTOX® DNA-binding dyes

flow cytometry histogram showing two distinct fluorescence intensity peaks for live and dead cells along with an additional scatter plot showing subpopulations within the live-cell group  

 

Figure 1. Viable cell gating with SYTOX® Dead Cell Stains. A mixture of heat-treated and untreated human peripheral blood leukocytes was stained with 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 SYTOX® Red fluorescence signal, and live cells, which do not. (B) Dual-parameter plot of CD8 and CD3 staining after gating on live cells.


Viability assays using classic cell-impermeant DNA-binding dyes

Cell-impermeant classic DNA-binding dyes include propidium iodide (Figure 2) and 7-AAD. Both of these dyes have been used extensively for viability assays in flow cytometry, since they can be excited by the 488 or 532 nm laser and emit at wavelengths beyond 610 nm.

Selection guide for classic cell-impermeant DNA-binding dyes

flow cytometry scatter plot showing three populations: viable, apoptotic, and necrotic  
Figure 2. Dead cell staining using propidium iodide. Jurkat cells were treated with 10 μM camptothecin for 4 hours and subsequently stained using the Dead Cell Apoptosis Kit with Annexin V Alexa Fluor® 488 & Propidium Iodide. Cells were analyzed by flow cytometry using 488 nm excitation on the Attune® Acoustic Focusing Cytometer with 530/30 and 575/24 bandpass filters and collected at a standard 100 μL/min collection rate. Dead (necrotic) cells display high levels of propidium iodide staining compared to apoptotic or viable cells. A=apoptotic cells, V=viable cells, N=necrotic cells.

Viability assays using esterase substrates

CellTrace™ Calcein AM dyes can be passively loaded into adherent and nonadherent cells. These cell-permeant esterase substrates serve as viability probes that measure both enzymatic activity, which is required to activate their fluorescence, and cell membrane integrity, which is required for intracellular retention of their fluorescent products. Available with blue, violet, and green fluorescence, these dyes are ideal for short-term staining of live cells and can be used in multiplexed flow cytometry experiments (Figure 3).

Selection guide for esterase substrates

flow cytometry scatter plot showing two distinct populations: dead cells (red fluorescent) and live cells (green fluorescent)  
Figure 3. CellTrace™ Calcein Green AM staining of live cells. A 1:1 mixture of live and ethanol-fixed (dead) human B cells was stained with calcein green AM and ethidium homodimer-1. After 5 minutes, flow cytometric analysis was carried out with excitation at 488 nm. The resulting bivariate frequency distribution shows the clear separation of the green-fluorescent (530 nm) live cell population from the red-fluorescent (585 nm) dead cell population.

Viability assays using esterase substrates and amine-reactive dyes

LIVE/DEAD® Violet Viability/Vitality Kit

The LIVE/DEAD® Violet Viability/Vitality Kit provides a two-color fluorescence cell viability and vitality assay based on the simultaneous determination of live and dead cells with two probes that measure recognized parameters of cell health: plasma membrane integrity as a measure of cell viability, and intracellular esterase activity as a measure of cell vitality (Figure 4). CellTrace™ Calcein Violet AM and LIVE/DEAD® Fixable Aqua fluorescent reactive dye are optimal dyes for this application; both dyes utilize the violet laser, allowing other laser lines to be used with more conventional markers.

See details for they LIVE/DEAD® Viability/Vitality Kit

flow cytometry scatter plot showing two distinct populations: live cells (stained with calcein) and dead cells (stained with aqua fluorescent dye)  
Figure 4. 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).
Selection guides for nonfixable viability dyes for flow cytometry
  SYTOX® Blue Dead Cell Stain SYTOX® Green Dead Cell Stain SYTOX® Orange Dead Cell Stain SYTOX® Red Dead Cell Stain SYTOX® Dead Cell Stain Sampler Pack
Basis of assay SYTOX® Dead Cell Stains enter cells with compromised cell membranes and bind to dsDNA/RNA. The dyes are mostly nonfluorescent until they bind to nucleic acids. Cannot pass through intact cell membranes and are not fixable. May also be used for DNA content cell cycle analysis in fixed cells but does require the addition of RNase. Allows exclusion of dead cells from flow cytometric analysis.
Readout Dead cells are highly fluorescent; live cells not stained
Laser (nm)
450
488
488
532
633/635
Various
Ex/Em (nm)
444/480
504/523
547/570
640/658
Various
Bibliography Citations
Multiplexable
Yes
Yes
Yes
Yes
Yes
Dye compatibility Cannot be used in combination with Pacific Blue™ dye, CellTrace™ Violet stain, FxCycle™ Violet stain, LIVE/DEAD® Fixable Violet Stain, BV421 or eFluor® 450 Cannot be used in combination with FITC, Alexa Fluor® 488, GFP, CellTrace™ CSFE stains or LIVE/DEAD® Fixable Green Stain Cannot be used in combination with R-PE, propidium iodide, SYTOX® AADvanced stain, 7-AAD, PE-Alexa Fluor® 610 and PE-Texas Red® tandem dyes Cannot be used in combination with APC, Alexa Fluor® 647, FxCycle™ Far Red, CellTrace™ Far Red stains
Fixable
No
No
No
No
No
Sample type
Live cells
Format
1,000 tests
1,000 tests
1,000 tests
1,000 tests
50 tests per stain
Cat. No.
  Propidium iodide 7-AAD (7-aminoactinomycin D)
Basis of assay Classic DNA binding dead cell dyes enter cells with compromised cell membranes and bind to dsDNA/RNA. Cannot pass through intact cell membranes and are not fixable. May also be used for DNA content cell cycle analysis in fixed cells but this requires the addition of RNase. Allows exclusion of dead cells from flow cytometric analysis.
Readout Dead cells are highly fluorescent; live cells are not stained
Laser (nm)
488
532
488
532
Ex/Em (nm)
535/617
546/647
Bibliography
Citations
Citations
Multiplexable
Yes
Yes
Dye compatibility Cannot be used in combination with R-PE, SYTOX® Orange Dead cell stain, SYTOX® AADvanced stain, 7-AAD, PE-Alexa Fluor® 610, or PE-Texas Red® tandem dyes Cannot be used in combination with R-PE, SYTOX® Orange Dead cell stain, SYTOX® AADvanced stain, 7-AAD, PE-Alexa Fluor® 610, or PE-Texas Red® tandem dyes
Fixable
No
No
Sample type
Live cells
Format
100 mg
100 mg
Cat. No.
  CellTrace™ Calcein Blue, AM CellTrace™ Calcein Violet, AM CellTrace™ Calcein Green, AM
Basis of assay CellTrace™ calcein AM dyes are nonfluorescent when the AM ester is intact and they cross membranes of all cells. Once inside live cells, the active esterases cleave the AM group which allows the dye to fluoresce. The AM group is not cleaved in dead cells and the dyes do not fluoresce. Best use is with short-term labeling as the dye, once the AM group is cleaved, can be actively transported out of the cell within a few hours.
Readout Live cells are highly fluorescent; dead cells display minimal fluorescence
Laser (nm)
UV
405
488
Ex/Em (nm)
322/425
400/452
495/515
Bibliography
Citations
Citations
Citations
Multiplexable
Yes
Yes
Yes
Dye compatibility Cannot be used in combination with Indo-1 (Blue), DAPI, or Hoechst 33342 Cannot be used in combination with Pacific Blue™ dye, CellTrace™ Violet stain, FxCycle™ Violet stain, LIVE/DEAD® Fixable Violet Stain, BV421, or eFluor® 450 Cannot be used in combination with FITC, Alexa Fluor® 488, GFP, or CellTrace™ CSFE stain
Fixable
No
No
No
Sample type
Live cells
Format
20 x 50 µg
20 x 50 µg
20 x 50 µg
Cat. No.
 
Basis of assay The LIVE/DEAD® Violet Viability/Vitality Kit provides a two-color fluorescence cell viability and vitality assay that is based on the simultaneous determination of live and dead cells with two probes that measure recognized parameters of cell health: plasma membrane integrity as a measure of cell viability, and intracellular esterase activity as a measure of cell vitality.
Fluorescent label Calcein Violet, AM LIVE/DEAD® Aqua stain
Readout Live cells are highly fluorescent; dead cells are significantly dimmer Live cells are minimally fluorescent; dead cells are highly fluorescent
Laser (nm)
405
405
Ex/Em (nm)
400/452
367/526
Multiplexable
Yes
Yes
Dye compatibility Cannot be used in combination with Pacific Blue™ dye, CellTrace™ Violet stain, FxCycle™ Violet stain, LIVE/DEAD® Fixable Violet Stain, BV421, or eFluor® 450 Cannot be used in combination with Pacific Green™ dye, F2N12S apoptosis assay, AmCyan, or BV510
Fixable
No
Sample type
Live cells
Format
20 tests
Cat. No.