section of flow cytometry histogram showing two distinct fluorescence peaks  

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 LIVE/DEAD® Fixable Dead Cell Stains are fixable viability dyes that help to ensure accurate assessment of cell viability in samples after fixation and/or permeabilization.

View selection guide for fixable viability dyes

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

  • Flexible—eight single-channel colors available for UV, 405, 488, 532, 561, or 633 nm lasers in three packaging sizes to match your experiment
  • Specific—live and dead cells are clearly differentiated, even after fixation, allowing easy exclusion of dead cells that can impact the accuracy of your results
  • Simple—fit into almost any staining and immunophenotyping protocol

See also viability assays for fluorescence imaging and microplates

LIVE/DEAD® Fixable Dead Cell Stains

LIVE/DEAD® Fixable Dead Cell 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 more intense staining (Figure 1, DEAD).

artist's rendition of two cells following use of the LIVE/DEAD stain; a nonfluorescent live cell next to a fluorescent dead cell  


Figure 1. Principle of the LIVE/DEAD® Fixable 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).

two flow cytometry histograms showing no change in the fluorescence intensity peaks detected in the unfixed and the fixed cell samples

Figure 2. Retention of LIVE/DEAD® Fixable Dead Cell Stains after fixation. The LIVE/ DEAD® Fixable Aqua Dead Cell Stain Kit 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 405 nm excitation and ~525 nm emission.

Selection guide for fixable viability dyes

  LIVE/DEAD® Fixable Blue stain LIVE/DEAD® Fixable Violet stain LIVE/DEAD® Fixable Aqua stain LIVE/DEAD® Fixable Yellow stain
Basis of assay LIVE/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.
Readout Dead cells are highly fluorescent and easy to distinguish from live cells, which are stained with minimal fluroescence
Laser (nm)
UV
405
405
405
Ex/Em (nm)
350/450
416/451
367/526
400/575
Bibliography
Citations
Multiplexable
Yes
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,  BV421 or eFluor® 450 Cannot be used in combination with Pacific Green™ dye, F2N12S apoptosis assay, AmCyan, or BV510 Cannot be used in combination with Pacific Orange™ dye, Qdot® 605, F2N12S apoptosis assay, or BV605
Fixable
Yes
Yes
Yes
Yes
Sample type
Live cells
Cat. No. 80 tests
200 tests
400 tests
  LIVE/DEAD® Fixable Green stain LIVE/DEAD® Fixable Red stain
Basis of assay LIVE/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.
Readout Dead cells are highly fluorescent and easy to distinguish from live cells, which are stained with minimal fluorescence
Laser (nm)
488
488
561
Ex/Em (nm)
495/520
595/615
Bibliography
Citations
Multiplexable
Yes
Yes
Dye compatibility Cannot be used in combination with FITC, Alexa Fluor® 488, GFP, or CellTrace® CSFE stain Cannot be used in combination with R-PE, propidium iodide, SYTOX® AADvanced stain, 7-AAD, PE-Alexa Fluor® 610 and PE-Texas Red® tandems
Fixable
Yes
Yes
Sample type
Live cells
Cat. No. 80 tests
200 tests
400 tests
  LIVE/DEAD® Fixable Far Red stain LIVE/DEAD® Fixable Near-IR stain
Basis of assay LIVE/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.
Readout Dead cells are highly fluorescent and easy to distinguish from live cells, which are stained with minimal fluorescence
Laser (nm)
633/635
633/635
Ex/Em (nm)
650/665
750/775
Bibliography
Citations
Multiplexable
Yes
Yes
Dye compatibility Cannot be used in combination with APC, Alexa Fluor® 647 dye, FxCycle™ Far Red and CellTrace® Far Red stains Cannot be used in combination with APC-Alexa Fluor® 750, APC-Cy® 7 or Vybrant® DyeCycle™ Ruby stain
Fixable
Yes
Yes
Sample type
Live cells
Cat. No. 80 tests
L34973
L34975
200 tests
L10120
L10119
400 tests
L34974
L34976

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 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