Cells stained with LIVE/DEAD aqua

Easily, reliably and quantitatively analyze live and dead cells within minutes

The ability to stain live cells with a viability dye and preserve that staining pattern after fixation is critical for intracellular immunophenotyping. Exclusion of the dead cells from the data allows cleaner separation and identification of cell populations. The Invitrogen LIVE/DEAD Fixable Viability Dyes are fixable viability dyes that help to ensure accurate assessment of cell viability in samples after fixation and/or permeabilization.

These flow cytometry–based kits provide you with tools that are:

  • Flexible—14 different LIVE/DEAD dyes excited from UV, 405, 488, 532, 561, 633, or 808 nm lasers and emission choices to different channels
  • Robust—clear distinction of live and dead cells is preserved for up to 30 days after fixation
  • Simple—fit into almost any staining and immunophenotyping protocol
  • Unique14 novel spectral signatures

See also viability assays for fluorescence imaging and microplates

View selection guide for fixable viability dyes


Introduction to LIVE/DEAD fixable technology

LIVE/DEAD Fixable Viability Stain Kits are based on the reaction of a fluorescent reactive dye with cellular proteins (amines). These dyes cannot penetrate live cell membranes, so only cell surface proteins are available to react with the dye, resulting in dim staining (Figure 1, LIVE). The reactive dye can permeate the damaged membranes of dead cells and stain both the interior and exterior amines, resulting in more intense staining (Figure 1, DEAD).

Artist image of LIVE/DEAD dye with a viable and dead cell

Figure 1. Principle of the LIVE/DEAD Fixable Viability Dead Cell Stains.The cell-impermeant, amine-reactive dye only binds to the surface of the live cell, resulting in very dim fluorescence. The dye can penetrate the cell membrane in dead cells and will bind to internal proteins, resulting in very bright fluorescence.

The difference in fluorescence intensity between the live and dead cell populations is typically greater than 50-fold, thereby allowing complete, simultaneous discrimination between the two cell populations (Figure 2A). Additionally, the covalent attachment of the dyes to proteins allows preservation of discrimination of the live and dead cell populations following fixation with formaldehyde, commonly used for intracellular immunophenotyping and to inactivate pathogens (Figure 2B). In contrast, non-fixable dyes including propidium iodide (PI) (Figure 3) lose staining pattern after fixation
(Figure 3B).

Histogram plot of cells stained with LIVE/DEAD aqua before or after fixation.

Figure 2. 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.

Histogram plot of cells stained with propidium iodide before or after fixation.

Figure 3. Cells stained with PI does not retain staining pattern after fixation. Propidium iodide (PI) 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 ~617 nm emission.

How to use LIVE/DEAD dyes with other fluorophores

Multi-step diagram of staining

Step 1: Stain cells with LIVE/DEAD reagent
Protocol for LIVE/DEAD cell staining.
Minimize FBS or BSA or protein buffer to <1%

Step 2: Use a fixation and/or permeabilization buffer kit if staining with intracellular markers or to persevere stained cells. Do not use a fixation agent before the LIVE/DEAD reagent.

Step 3: Stain cells with flow cytometry antibodies.
Combinations of dyes and clones, and reagents can be created on the Invitrogen Flow Cytometry Panel Builder.
Protocol for intracellular staining for flow cytometry.


Gating for viable and dead cells

Figure 5. Live cells will have less fluorescence since LIVE/DEAD dye will bind to cell surface proteins. Dead cells will be the far-right population and exhibit bright signal as LIVE/DEAD dye goes through damaged cell membranes and binds to internal proteins. Middle population is structural degradation of cells and will incorporate less dye than dead cells. Jurkat cells were incubated with LIVE/DEAD Fixable Lime (506) Viability kit for 405 nm excitation for 20 mins and then fixed. Dead cells were induced by heat treatment.

Figure 6. Histograms of Jurkat cells stained with different LIVE/DEAD viability dyes. Dead cell population was created with heat treatment. Cell samples were stained for 20 min with LIVE/DEAD fixable stains.

Selection guide for fixable viability dyes

 LIVE/DEAD Fixable Blue stainLIVE/DEAD
Fixable Violet stain
LIVE/DEAD Fixable Lime stainLIVE/DEAD Fixable Aqua stainLIVE/DEAD Fixable Yellow stain
Basis of assayLIVE/DEAD Fixable Dead Cell Stain kits are fixable viability dyes that distinguish live cells from dead cells based on cell membrane integrity and access to available amines. Cells can then be fixed for intracellular antigen detection without loss of original cell staining pattern. Allows easy exclusion of dead cells from flow cytometry readout.
ReadoutDead cells are highly fluorescent and easy to distinguish from live cells, which are stained with minimal fluorescence
Laser (nm)
UV
405
405
405
405
Ex/Em (nm)
350/450
416/451
405/506
367/526
400/575
Bibliography
Citations
Multiplexable
Yes
Yes
Yes
Yes
Yes
Dye compatibilityCannot be used in combination with Invitrogen product line such as Indo-1 (blue), DAPI, or Hoechst 33342Cannot be used in combination with Invitrogen product line such as Pacific Blue dye, CellTrace Violet stain, FxCycle Violet stain,  BV421 or eFluor 450Cannot be used in combination with Invitrogen product line such as eFluor 506 and Pacific orangeCannot be used in combination with Invitrogen product line such as Pacific Green dye, F2N12S apoptosis assay, AmCyan, or BV510Cannot be used in combination with Invitrogen product line such as Pacific Orange dye, Qdot 605, F2N12S apoptosis assay, or BV605
Fixable
Yes
Yes
Yes
Yes
Yes
Sample type
Live cells
Cat. No.80 testsL34989
200 testsL34990
400 testsL34991
 LIVE/DEAD Fixable Green stainLIVE/DEAD Fixable Olive stainLIVE/DEAD Fixable Orange stainLIVE/DEAD Fixable Red stain
Basis of assayLIVE/DEAD Fixable Dead Cell Stain kits are fixable viability dyes that distinguish live cells from dead cells based on cell membrane integrity and access to available amines. Cells can then be fixed for intracellular antigen detection without loss of original cell staining pattern. Allows easy exclusion of dead cells from flow cytometry readout.
ReadoutDead cells are highly fluorescent and easy to distinguish from live cells, which are stained with minimal fluorescence
Laser (nm)
488
488
561
488
561
Ex/Em (nm)
495/520
480/557
580/602
595/615
Bibliography
Citations
Multiplexable
Yes
YesYes
Yes
Dye compatibilityCannot be used in combination with Invitrogen product line such as FITC, Alexa Fluor 488, GFP, or CellTrace CSFE stainCannot be used in combination with Invitrogen product line such as FITC, Alexa Fluor 488, Nova Fluor Blue 510, GFP, LIVE/DEAD Fixable Green stain, or CellTrace CSFE stainCannot be used in combination with Invitrogen product line such as PE-Alexa Fluor 610 PE-eFluor 610, PE-Texas Red, and NovaFluor Blue 610Cannot be used in combination with Invitrogen product line such as R-PE, propidium iodide, SYTOX AADvanced stain, 7-AAD, PE-Alexa Fluor 610 and PE-Texas Red tandems
Fixable
Yes
YesYes
Yes
Sample type
Live cells
Cat. No.80 testsL34977L34983
200 testsL34978L34984
400 testsL34979L34985
 LIVE/DEAD Fixable Far Red stainLIVE/DEAD Fixable Scarlet stainLIVE/DEAD Fixable Near-IR (775) stainLIVE/DEAD Fixable Near-IR (780) stain
Basis of assayLIVE/DEAD Fixable Dead Cell Stain kits are fixable viability dyes that distinguish live cells from dead cells based on cell membrane integrity and access to available amines. Cells can then be fixed for intracellular antigen detection without loss of original cell staining pattern. Allows easy exclusion of dead cells from flow cytometry readout.
ReadoutDead cells are highly fluorescent and easy to distinguish from live cells, which are stained with minimal fluorescence
Laser (nm)
633/635
633/635
633/635
633/780
Ex/Em (nm)
650/665
702/723
750/775
633/785
Bibliography
Multiplexable
Yes
Yes Yes
Yes
Dye compatibilityCannot be used in combination with Invitrogen product line such as APC, Alexa Fluor 647 dye, FxCycle Far Red and CellTrace Far Red stainsCannot be used in combination with Invitrogen product line such as Alexa Fluor 700, APC-Cyanine 5.5, NovaFluor Red 700, and DRAQ5Cannot be used in combination with Invitrogen product line such as APC-Alexa Fluor 750, APC-Cy 7 or Vybrant DyeCycle Ruby stainCannot be used in combination with Invitrogen product line such as APC-Alexa Fluor 750, APC-eFluor 780
FixableYesYesYesYes
Sample type
Live cells
Cat. No.80 tests
L34973
L34986

L34975

L34992
200 tests
L10120
L34987
L10119
L34993
400 tests
L34974
L34988

L34976

L34994
 LIVE/DEAD Fixable Near IR (876) stain
Basis of assayLIVE/DEAD Fixable Dead Cell Stain kits are fixable viability dyes that distinguish live cells from dead cells based on cell membrane integrity and access to available amines. Cells can then be fixed for intracellular antigen detection without loss of original cell staining pattern. Allows easy exclusion of dead cells from flow cytometry readout.
ReadoutDead cells are highly fluorescent and easy to distinguish from live cells, which are stained with minimal fluorescence
Laser (nm)
808
Ex/Em (nm)
840/876
Multiplexable
Yes
Dye compatibility
Fixable
Yes
Sample type
Live cells
Cat. No.80 tests
200 tests
400 tests
 LIVE/DEAD Fixable Blue stainLIVE/DEAD
Fixable Violet stain
LIVE/DEAD Fixable Lime stainLIVE/DEAD Fixable Aqua stainLIVE/DEAD Fixable Yellow stain
Basis of assayLIVE/DEAD Fixable Dead Cell Stain kits are fixable viability dyes that distinguish live cells from dead cells based on cell membrane integrity and access to available amines. Cells can then be fixed for intracellular antigen detection without loss of original cell staining pattern. Allows easy exclusion of dead cells from flow cytometry readout.
ReadoutDead cells are highly fluorescent and easy to distinguish from live cells, which are stained with minimal fluorescence
Laser (nm)
UV
405
405
405
405
Ex/Em (nm)
350/450
416/451
405/506
367/526
400/575
Bibliography
Citations
Multiplexable
Yes
Yes
Yes
Yes
Yes
Dye compatibilityCannot be used in combination with Invitrogen product line such as Indo-1 (blue), DAPI, or Hoechst 33342Cannot be used in combination with Invitrogen product line such as Pacific Blue dye, CellTrace Violet stain, FxCycle Violet stain,  BV421 or eFluor 450Cannot be used in combination with Invitrogen product line such as eFluor 506 and Pacific orangeCannot be used in combination with Invitrogen product line such as Pacific Green dye, F2N12S apoptosis assay, AmCyan, or BV510Cannot be used in combination with Invitrogen product line such as Pacific Orange dye, Qdot 605, F2N12S apoptosis assay, or BV605
Fixable
Yes
Yes
Yes
Yes
Yes
Sample type
Live cells
Cat. No.80 testsL34989
200 testsL34990
400 testsL34991
 LIVE/DEAD Fixable Green stainLIVE/DEAD Fixable Olive stainLIVE/DEAD Fixable Orange stainLIVE/DEAD Fixable Red stain
Basis of assayLIVE/DEAD Fixable Dead Cell Stain kits are fixable viability dyes that distinguish live cells from dead cells based on cell membrane integrity and access to available amines. Cells can then be fixed for intracellular antigen detection without loss of original cell staining pattern. Allows easy exclusion of dead cells from flow cytometry readout.
ReadoutDead cells are highly fluorescent and easy to distinguish from live cells, which are stained with minimal fluorescence
Laser (nm)
488
488
561
488
561
Ex/Em (nm)
495/520
480/557
580/602
595/615
Bibliography
Citations
Multiplexable
Yes
YesYes
Yes
Dye compatibilityCannot be used in combination with Invitrogen product line such as FITC, Alexa Fluor 488, GFP, or CellTrace CSFE stainCannot be used in combination with Invitrogen product line such as FITC, Alexa Fluor 488, Nova Fluor Blue 510, GFP, LIVE/DEAD Fixable Green stain, or CellTrace CSFE stainCannot be used in combination with Invitrogen product line such as PE-Alexa Fluor 610 PE-eFluor 610, PE-Texas Red, and NovaFluor Blue 610Cannot be used in combination with Invitrogen product line such as R-PE, propidium iodide, SYTOX AADvanced stain, 7-AAD, PE-Alexa Fluor 610 and PE-Texas Red tandems
Fixable
Yes
YesYes
Yes
Sample type
Live cells
Cat. No.80 testsL34977L34983
200 testsL34978L34984
400 testsL34979L34985
 LIVE/DEAD Fixable Far Red stainLIVE/DEAD Fixable Scarlet stainLIVE/DEAD Fixable Near-IR (775) stainLIVE/DEAD Fixable Near-IR (780) stain
Basis of assayLIVE/DEAD Fixable Dead Cell Stain kits are fixable viability dyes that distinguish live cells from dead cells based on cell membrane integrity and access to available amines. Cells can then be fixed for intracellular antigen detection without loss of original cell staining pattern. Allows easy exclusion of dead cells from flow cytometry readout.
ReadoutDead cells are highly fluorescent and easy to distinguish from live cells, which are stained with minimal fluorescence
Laser (nm)
633/635
633/635
633/635
633/780
Ex/Em (nm)
650/665
702/723
750/775
633/785
Bibliography
Multiplexable
Yes
Yes Yes
Yes
Dye compatibilityCannot be used in combination with Invitrogen product line such as APC, Alexa Fluor 647 dye, FxCycle Far Red and CellTrace Far Red stainsCannot be used in combination with Invitrogen product line such as Alexa Fluor 700, APC-Cyanine 5.5, NovaFluor Red 700, and DRAQ5Cannot be used in combination with Invitrogen product line such as APC-Alexa Fluor 750, APC-Cy 7 or Vybrant DyeCycle Ruby stainCannot be used in combination with Invitrogen product line such as APC-Alexa Fluor 750, APC-eFluor 780
FixableYesYesYesYes
Sample type
Live cells
Cat. No.80 tests
L34973
L34986

L34975

L34992
200 tests
L10120
L34987
L10119
L34993
400 tests
L34974
L34988

L34976

L34994
 LIVE/DEAD Fixable Near IR (876) stain
Basis of assayLIVE/DEAD Fixable Dead Cell Stain kits are fixable viability dyes that distinguish live cells from dead cells based on cell membrane integrity and access to available amines. Cells can then be fixed for intracellular antigen detection without loss of original cell staining pattern. Allows easy exclusion of dead cells from flow cytometry readout.
ReadoutDead cells are highly fluorescent and easy to distinguish from live cells, which are stained with minimal fluorescence
Laser (nm)
808
Ex/Em (nm)
840/876
Multiplexable
Yes
Dye compatibility
Fixable
Yes
Sample type
Live cells
Cat. No.80 tests
200 tests
400 tests


LIVE/DEAD Fixable Dead Cell Stain Sampler Kit

Not sure about which dye will suit your needs, or need even more flexibility in your experiments? Try our LIVE/DEAD Fixable Dead Cell Stain Sampler Kit, which contains enough of each color stain to run 40 tests.

Compensation for LIVE/DEAD Fixable Dead Cell Stains

The Invitrogen ArC Amine Reactive Compensation Bead Kit provides a consistent, accurate, and simple-to-use technique for the setting of flow cytometry compensation when using any of the LIVE/DEAD Fixable Dead Cell Stain Kits or any amine-reactive dye. Advantages of the kit include:

  • No need to heat-treat cells
  • Optimized for LIVE/DEAD Fixable Dead Cell Stain Kits
  • Fast and simple bead-based flow cytometry compensation
  • Precious samples are not needed for setting compensation
  • Accurate and consistent results
ArC Amine Reactive Compensation Bead Kit

Figure 3. Beads from the ArC Bead Kit were stained with LIVE/DEAD Fixable Violet Dead Cell Stain Kit (V34955) and analyzed by flow cytometry. A mixture of the positive and negative beads in the ArC Bead Kit (A 10346) were stained with LIVE/DEAD Fixable Violet Dead Cell Stain Kit (V34955), washed, and analyzed using a flow cytometry equipped with a 405/7nm laser with 450/50 nm band pass filter. Histogram shows positive and negative peaks clearly distinguishable.

   

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