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
- Unique—14 novel spectral signatures
See also viability assays for fluorescence imaging and microplates
View selection guide for fixable viability dyes
On demand webinar
New fixable viability dyes and applications for flow cytometry
This webinar will provide an overview of methods and reagents to assess cell viability with flow cytometry. Discussion will focus on our recent efforts to expand the color palette of 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).
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).
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.
Figure 3. Cells stained with PI do 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
Figure 4. LIVE/DEAD dyes have minimal spillover and allow for larger and more accurate flow cytometry experiments. Human PBMCs labeled with LIVE/DEAD Near IR (876) Viability kit, for 808 nm excitation (Cat. No. L34981), CD4 Alexa Fluor 647 (Cat. No. 51-0049-42) and CD8 APC-Cy7 (Cat. No. A15448). All excited by 808 nm laser.
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 stain | LIVE/DEAD
Fixable Violet stain | LIVE/DEAD Fixable Lime 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 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 compatibility | Cannot be used in combination with Invitrogen product line such as Indo-1 (blue), DAPI, or Hoechst 33342 | Cannot be used in combination with Invitrogen product line such as Pacific Blue dye, CellTrace Violet stain, FxCycle Violet stain, BV421 or eFluor 450 | Cannot be used in combination with Invitrogen product line such as eFluor 506 and Pacific orange | Cannot be used in combination with Invitrogen product line such as Pacific Green dye, F2N12S apoptosis assay, AmCyan, or BV510 | Cannot 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 tests | | | L34989 | | |
| 200 tests | | | L34990 | | |
| 400 tests | | | L34991 | | |
| | LIVE/DEAD Fixable Green stain | LIVE/DEAD Fixable Olive stain | LIVE/DEAD Fixable Orange 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 | 488 | 561 |
| Ex/Em (nm) | 495/520 | 480/557 | 580/602 | 595/615 |
| Bibliography |
Citations
|
| Multiplexable |
Yes
| Yes | Yes |
Yes
|
| Dye compatibility | Cannot be used with NB510, NB530, NB555 | Can be used with NB510, FITC, AF488. Cannot be used with NB555, NB585 | Can be used with NB610. Cannot be used with NY590, NY610 | Cannot be used with NY610, NY590 |
| Fixable |
Yes
| Yes | Yes |
Yes
|
| Sample type |
Live cells
|
| Cat. No. | 80 tests | | L34977 | L34983 | |
| 200 tests | | L34978 | L34984 | |
| 400 tests | | L34979 | L34985 | |
| | LIVE/DEAD Fixable Far Red stain | LIVE/DEAD Fixable Scarlet stain | LIVE/DEAD Fixable Near-IR (775) stain | LIVE/DEAD Fixable Near-IR (780) 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 | 633/635 | 633/780 |
| Ex/Em (nm) | 650/665 | 702/723 | 750/775 | 633/785 |
| Bibliography | |
| Multiplexable |
Yes
| Yes | Yes |
Yes
|
| Dye compatibility | Cannot be used with NR660 | Cannot be used in combination with Invitrogen product line such as Alexa Fluor 700, APC-Cyanine 5.5, NovaFluor Red 700, and DRAQ5 | Cannot be used in combination with Invitrogen product line such as APC-Alexa Fluor 750, APC-Cy 7 or Vybrant DyeCycle Ruby stain | Cannot be used in combination with Invitrogen product line such as APC-Alexa Fluor 750, APC-eFluor 780 |
| Fixable | Yes | Yes | Yes | Yes |
| 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 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) | 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 stain | LIVE/DEAD
Fixable Violet stain | LIVE/DEAD Fixable Lime 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 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 compatibility | Cannot be used in combination with Invitrogen product line such as Indo-1 (blue), DAPI, or Hoechst 33342 | Cannot be used in combination with Invitrogen product line such as Pacific Blue dye, CellTrace Violet stain, FxCycle Violet stain, BV421 or eFluor 450 | Cannot be used in combination with Invitrogen product line such as eFluor 506 and Pacific orange | Cannot be used in combination with Invitrogen product line such as Pacific Green dye, F2N12S apoptosis assay, AmCyan, or BV510 | Cannot 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 tests | | | L34989 | | |
| 200 tests | | | L34990 | | |
| 400 tests | | | L34991 | | |
| | LIVE/DEAD Fixable Green stain | LIVE/DEAD Fixable Olive stain | LIVE/DEAD Fixable Orange 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 | 488 | 561 |
| Ex/Em (nm) | 495/520 | 480/557 | 580/602 | 595/615 |
| Bibliography |
Citations
|
| Multiplexable |
Yes
| Yes | Yes |
Yes
|
| Dye compatibility | Cannot be used with NB510, NB530, NB555 | Can be used with NB510, FITC, AF488. Cannot be used with NB555, NB585 | Can be used with NB610. Cannot be used with NY590, NY610 | Cannot be used with NY610, NY590 |
| Fixable |
Yes
| Yes | Yes |
Yes
|
| Sample type |
Live cells
|
| Cat. No. | 80 tests | | L34977 | L34983 | |
| 200 tests | | L34978 | L34984 | |
| 400 tests | | L34979 | L34985 | |
| | LIVE/DEAD Fixable Far Red stain | LIVE/DEAD Fixable Scarlet stain | LIVE/DEAD Fixable Near-IR (775) stain | LIVE/DEAD Fixable Near-IR (780) 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 | 633/635 | 633/780 |
| Ex/Em (nm) | 650/665 | 702/723 | 750/775 | 633/785 |
| Bibliography | |
| Multiplexable |
Yes
| Yes | Yes |
Yes
|
| Dye compatibility | Cannot be used with NR660 | Cannot be used in combination with Invitrogen product line such as Alexa Fluor 700, APC-Cyanine 5.5, NovaFluor Red 700, and DRAQ5 | Cannot be used in combination with Invitrogen product line such as APC-Alexa Fluor 750, APC-Cy 7 or Vybrant DyeCycle Ruby stain | Cannot be used in combination with Invitrogen product line such as APC-Alexa Fluor 750, APC-eFluor 780 |
| Fixable | Yes | Yes | Yes | Yes |
| 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 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) | 808 |
| Ex/Em (nm) | 840/876 |
| Multiplexable |
Yes
|
| Dye compatibility | — |
| Fixable |
Yes
|
| Sample type |
Live cells
|
| Cat. No. | 80 tests | |
| 200 tests | |
| 400 tests | |
Figure 7. LIVE/DEAD fixable dye signatures generated on Cytek Aurora Spectral analyzer.
Figure 8. LIVE/DEAD fixable dye signatures generated on Invitrogen Bigfoot Spectral Cell Sorter.
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
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.
