Zombie Fixable Viability™ Sampler Kit

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Other Names
Fixable Dye (live/dead), Fixable Viability Dye,Dead Cell Stain Kit, Dead Cell Staining Kit
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A_Zombie_UV_and_Violet_Sampler_Kit_1_060917
One day old C57BL/6 mouse splenocytes were stained with Zombie dyes as indicated and analyzed before fixation (blue/purple) or after fixation and permeabilization (red). Cells alone, without Zombie staining, are indicated in black.
  • A_Zombie_UV_and_Violet_Sampler_Kit_1_060917
    One day old C57BL/6 mouse splenocytes were stained with Zombie dyes as indicated and analyzed before fixation (blue/purple) or after fixation and permeabilization (red). Cells alone, without Zombie staining, are indicated in black.
  • B_Zombie_Aqua_and_Yellow_Sampler_Kit_2_060917
    One day old C57BL/6 mouse splenocytes were stained with Zombie dyes as indicated and analyzed before fixation (blue/purple) or after fixation and permeabilization (red). Cells alone, without Zombie staining, are indicated in black.
  • C_Zombie_NIR_Sampler_Kit_3_060917
    One day old C57BL/6 mouse splenocytes were stained with Zombie dyes as indicated and analyzed before fixation (blue/purple) or after fixation and permeabilization (red). Cells alone, without Zombie staining, are indicated in black.
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423117 1 kit £202
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Description

The Zombie dyes in this kit (provided at 100 tests each) are the UV, NIR, Violet, Aqua and Yellow variants.

The Zombie live/dead fixable dyes are amine-reactive fluorescent dyes that are impermeant to live cells but permeant to cells with compromised membranes. They irreversibly conjugate to primary amine-containing proteins. Since there is a greater abundance of proteins inside the cell versus the cell surface, dead cells with a compromised membrane stain brighter than live cells with an intact membrane. Thus, they can be used to assess live vs. dead status of mammalian cells. However, titration of the reagent is required to ensure that live cells have minimal to no staining due to this dye on the cell type of interest. It is also important to consider that the accuracy of the live/dead assessment is dependent on the uniformity in size of each cell since larger cells will stain brighter than smaller cells, possibly confusing large cells as dead cells. Zombie live/dead fixable probes are useful for incorporation into multicolor panels, particularly in applications where cells will be fixed and permeabilized prior to analysis.

To find the spectra of each of the Zombie dyes offered in this kit, please find them here:  https://www.biolegend.com/spectraanalyzer

Product Details
Technical Data Sheet (pdf)

Product Details

Preparation
Zombie Fixable Viability Kit is composed of 5 vials of 100 tests of each lyophilized Zombie dye and anhydrous DMSO. For reconstitution, bring the kit to room temperature only when ready to use the reagent; add 100 µl of DMSO to each vial of Zombie dye until fully dissolved.
Storage & Handling
Store kit at -20°C upon receipt. Do not open vials until needed. Once the DMSO is added to the Zombie dye, use immediately, or store at -20°C in a dry place and protected from light, preferably in a desiccator or in a container with desiccant for no more than one month. The unopened kit is guaranteed for six months.

This product has a shelf-life of 12 months or less. Please contact technical support for lot specific CoA and expiration date inquiries of this product.
Application

FC - Quality tested
ICFC, IF - Validated

Recommended Usage

Each lot of this reagent is quality control tested by immunofluorescent staining with flow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is 1:100-1:1000 dilution per 1-10 million cells in 100 µl volume. It is recommended that the reagent be titrated for optimal performance for each application. For immunofluorescence microscopy, the suggested dilution is 1:1000. It is recommended that the reagent be titrated for optimal performance for each application, as optimal dosage varies with cell type.

Application Notes

Standard Cell Staining Protocol:

  1. Prior to reconstitution, spin down the vial of lyophilized reagent in a microcentrofuge to ensure the reagent is at the bottom of the vial.
  2. For reconstitution, pre-warm the kit to room temperature; add 100 µl of DMSO to one vial of Zombie dye and mix until fully dissolved
  3. Wash cells with PBS buffer (no Tris buffer and protein free).
  4. Dilute Zombie dye at 1:100-1000 in PBS. Resuspend 1-10 x 106 cells in diluted 100 µl Zombie solution. To minimize background staining of live cells, titrate the amount of dye and/or number of cells per 100 µl for optimal performance. Different cell types can have a wide degree of variability in staining based on cell size and degree of cell death.

    Note: Don’t use Tris buffer as a diluent and be sure that the PBS does not contain any other protein like BSA or FBS.
    Note: The amount of dye used can also influence the ability to detect apoptotic as well as live and dead cells.

  5. Incubate the cells at room temperature, in the dark, for 15-30 minutes.
  6. Wash one time with 2 ml BioLegend’s Cell Staining Buffer (Cat. No. 420201) or equivalent buffer containing serum or BSA.
  7. Continue performing antibody staining procedure as desired.
  8. Cells can be fixed with paraformaldehyde or methanol prior to permeabilization or can be analyzed without fixation.

 

No-wash Sequential Staining Protocol:

 

  1. Wash cells with PBS buffer (no Tris buffer and protein free).
  2. For reconstitution, pre-warm the kit to room temperature; add 100 µl of DMSO to one vial of Zombie dye and mix until fully  dissolved
  3. Determine the total µl volume of antibody cocktail previously titrated and optimized for the assay that will be added to each vial/well of cells based on a final volume of 100 µl. Subtract that antibody volume from the 100 µl total staining volume intended for the assay. In the remaining volume, dilute Zombie dye at 1:100-1000 in PBS as determined by prior optimization at that volume. For example, if you are adding 20 µl of antibody cocktail for a 100 µl total staining volume, use 80 µl of Zombie solution. Resuspend 1-10 x 106 cells in the appropriate volume of Zombie solution. Different cell types can have a wide degree of variability in staining based on cell size and degree of cell death.

    Note: Don’t use Tris buffer as a diluent and be sure that the PBS does not contain any other protein like BSA or FBS.
    Note: The amount of dye used can also influence the ability to detect apoptotic as well as live and dead cells.

  4. Incubate for 10-15 minutes at RT, protected from light. Without washing the cells, add the cell surface antibody cocktail and incubate for another 15-20 minutes.
  5. Add 1-2 mL Cell Staining Buffer (Cat. No. 420201) or equivalent buffer containing BSA or serum. Centrifuge to pellet.
  6. Continue with normal fixation and permeabilization procedure. If planning to skip fixation and analyze cells live, complete an additional wash step to minimize any unnecessary background of the live cells.


Notes: If the cell type in use cannot tolerate a protein-free environment, then titrate the Zombie dye in the presence of the same amount of BSA/serum as will be present in the antibody staining procedure. A higher amount of Zombie dye may be required since the BSA/serum will react with and bind up some proportion of the Zombie dye.

Antigen Details

Biology Area
Apoptosis/Tumor Suppressors/Cell Death, Cell Biology, Neuroscience
Gene ID
NA

Related FAQs

Can I use methanol/ethanol for fixation after using a Zombie dye?

Yes, most fixation reagents are fine to be used with Zombie dyes. However, it should be noted that Zombie dyes can still be sensitive to reactive oxygen species. Light exposure or reagents with hydrogen peroxide can lead to free radical formation, affecting fluorescence.

Can I use your Zombie dyes for microscopy application?

Zombie dyes that have been tested for microscopy applications in-house will display data on the product webpage. It should be noted that Zombie may not work for dead cell discrimination in every microscopy application, as a complicated point will be to determine the level of Zombie signal that constitutes a dead cell. Another difficulty may be finding the proper plane for microscopy in order to observe the dead cells.

Can I use Zombie and Annexin V to detect apoptotic cells?

Yes, Zombie can be used with Annexin V to discriminate live, apoptotic, and dead cells. Cells double positive for both Zombie and Annexin V are dead, while Zombie-dim/Annexin V-positive cells are apoptotic. Live cells will be Zombie-low and Annexin V-negative. The advantage to Zombie over PI and 7-AAD is that you can now fix and/or permeabilize the cells to stain for cell surface and intracellular antigens.

Can I use Zombie with cells suspension containing serum?
Serum is full of proteins which will sequester the dye and thereby reducing its effective concentration. The basic rule of thumb with zombie is to titrate it based on your specific condition. Titration also helps reduce the background and spillover into other channels.
Can Zombie be used to determine bacteria, yeast viability?
We have not tested in house bacterial or yeast viability using Zombie dyes. It is not clear whether the difference between surface and intracellular signals will be significantly different in case of non mammalian cells.
How does the performance of your Zombie dye compare with competitors?

Zombie dyes have been tested against other leading competitors' fixable viability kits and given comparable results. We also highly recommend that you titrate down the amount of each dye used in order to best match the negative signals of your unstained sample and MFI- (mean fluorescence intensity) stained samples.

I am concerned about the spillover I am observing from the Zombie dye into its neighboring channels.
Rule of thumb with Zombie dyes is to titrate them down as much as possible to fit your application. This should potentially help with spillover. Secondly, Zombie positive events represent dead cells and are typically gated out from analysis.
Why can't I fix my cells prior to using Zombie?

The fixation process can contort and alter the membrane of cells, effectively rendering them as dead. Since the ability of Zombie to stain dead cells is correlated with cell permeability, your results may no longer be a valid representation of dead versus live cells.

Go To Top Version: 2    Revision Date: 05/30/2019

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*These products may be covered by one or more Limited Use Label Licenses (see the BioLegend Catalog or our website, www.biolegend.com/ordering#license). BioLegend products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products, reverse engineer functionally similar materials, or to provide a service to third parties without written approval of BioLegend. By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. Unless otherwise indicated, these products are for research use only and are not intended for human or animal diagnostic, therapeutic or commercial use.

 

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