Zombie Red™ Fixable Viability Kit

Pricing & Availability
Regulatory Status
RUO
Other Names
Fixable Dye, Fixable Viability Dye
Ave. Rating
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Product Citations
publications
a-Zombie_Red_Viability_Dye_012714
One day old C57BL/6 mouse splenocytes were stained with Zombie Red™ and analyzed before fixation (purple) or after fixation and permeabilization (red). Cells alone, without Zombie Red™ staining, are indicated in black.
  • a-Zombie_Red_Viability_Dye_012714
    One day old C57BL/6 mouse splenocytes were stained with Zombie Red™ and analyzed before fixation (purple) or after fixation and permeabilization (red). Cells alone, without Zombie Red™ staining, are indicated in black.
  • b-Zombie_Red_Kit_ICC_012721
    HeLa cells were treated with 20% EtOH for 20 seconds, washed twice with PBS, and then were left to recover for five minutes with cell culture media in 37°C. The cells were stained with Zombie Red™ (1:1000) (red) for 15 minutes and then fixed with 1% paraformaldehyde (PFA) for ten minutes. Nuclei were counterstained with DAPI (blue) for five minutes. The image was captured with 40X objective.
See Zombie Red™ spectral data See high resolution IF data...
Cat # Size Price Quantity Avail. Save
423109 100 tests £51.30
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423110 500 tests £194.94
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Description

Zombie Red™ is an amine reactive fluorescent dye that is non-permeant to live cells, but permeant to the cells with compromised membranes. Thus, it can be used to assess live vs. dead status of mammalian cells. Zombie Red™ is a polar water soluble dye, providing red fluorescence, making it suitable for multi-color detection.

Product Details
Technical Data Sheet (pdf)

Product Details

Preparation
Zombie Red™ Fixable Viability kit is composed of lyophilized Zombie Red™ dye and anhydrous DMSO. For reconstitution, bring the kit to room temperature; add 100 µl of DMSO to one vial of Zombie Red™ dye until fully dissolved. 100 tests = 1 vial of Zombie Red™ + DMSO, 500 tests = 5 vials of Zombie Red™ + DMSO.
Storage & Handling
Store kit at -20°C upon receipt. Do not open vials until needed. Once the DMSO is added to the Zombie Red™ 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.
Application

FC, ICFC - Quality tested
ICC - Verified

Recommended Usage

Each lot of this product is quality control tested by immunofluorescent staining with flow cytometric analysis. For flow cytometry, the suggested dilution is 1:100-1:1000 for 1-10 million cells. For immunocytochemistry, 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.

Excitation Laser
Blue Laser (488 nm)
Green Laser (532 nm)/Yellow-Green Laser (561 nm)
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 Red™ dye and mix until fully dissolved
  3. Wash cells with PBS buffer (no Tris buffer and protein free).
  4. Dilute Zombie Red™ dye at 1:100-1000 in PBS. Resuspend 1-10 x 106 cells in diluted 100 µl Zombie Red™ 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 Red™ 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 Red™ 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 Red™ solution. Resuspend 1-10 x 106 cells in the appropriate volume of Zombie Red™ 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 Red™ 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 Red™ may be required since the BSA/serum will react with and bind up some proportion of the Zombie Red™.

Zombie Red™ dye is excited by the yellow/green laser (561 nm) and has fluorescence emission maximum at 624 nm. If using in a multi-color panel design, filter optimization may be required depending on other fluorophores used. Zombie Red™ dye has similar emission to PE-Texas Red.

Zombie Red™ can be weakly excited by the 488 laser, if a yellow/green laser is not available.  This may require a higher concentration of Zombie Red™ dye; optimization by the end user would be required.

Product Citations
  1. Mercer F, et al. 2016. PLoS Negl Trop Dis. 10: 0004913. PubMed
  2. Martin C, et al. 2017. PLoS One. 10.1371/journal.pone.0170504. PubMed
  3. Antsiferova M, et al. 2017. EMBO Mol Med. 9(1):27-45. PubMed
  4. Holokai L, et al. 2020. Cancers (Basel). 12:00. PubMed
  5. Krausgruber T, et al. 2020. Nature. 583:296. PubMed
  6. Lindner SE, et al. 2020. Immunohorizons. 0.356944444. PubMed
  7. Feng J,et al. 2017. Nat Commun. . 10.1038/s41467-017-01056-8. PubMed
  8. Riopel M, et al. 2019. Mol Metab. 20:89. PubMed
  9. Sperber HS, et al. 2019. Sci Rep. 9:707. PubMed
  10. Yen WF et al. 2019. Cell reports. 27(5):1472-1486 . PubMed
  11. Brennan FH, et al. 2019. JCI Insight. 4. PubMed
  12. Haertel E, et al. 2018. Eur J Immunol. 48:1001. PubMed
  13. Mousseau G, et al. 2019. MBio. 10. PubMed
  14. Ligorio M, et al. 2019. Cell. 178:160. PubMed

Antigen Details

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

Related FAQs

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.
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.

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 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.
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 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.

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.

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.

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DAPI (4',6-Diamidino-2-Phenylindole, Dilactate) ICFC, ICC
Rainbow Fluorescent Particles, 1 peak (3.0-3.4 µm) - Mid Range Intensity FC
Rainbow Calibration Particles, 8 peaks (3.0-3.4 µm) FC
Rainbow Calibration Particles, 6 peaks (3.0-3.4 µm) FC
Rainbow Fluorescent Particles, 1 peak (3.0-3.4 µm) - Bright Intensity FC
Cyto-Last™ Buffer ICFC
Recombinant Human TSLP (carrier-free) BA
Recombinant Mouse CXCL2 (MIP-2) (carrier-free) BA
Recombinant Human CCL1 (I-309) (carrier-free) BA
ELISA MAX™ Deluxe Set Mouse IL-10 ELISA
ELISA MAX™ Standard Set Mouse IL-10 ELISA
Recombinant Mouse CCL11 (Eotaxin) (carrier-free) BA
Recombinant Human CCL11 (Eotaxin) (carrier-free) BA
Recombinant Mouse VEGF-164 (carrier-free) BA
Recombinant Human VEGF-121 (carrier-free) BA
Recombinant Human TGF-β2 (carrier-free) BA
LEGEND MAX™ Mouse OVA Specific IgE ELISA Kit ELISA
LEGEND MAX™ Human IL-34 ELISA Kit ELISA
ELISA MAX™ Deluxe Set Human IL-34 ELISA
Brilliant Violet 510™ Streptavidin FC, ICFC
APC Annexin V FC
PE Annexin V FC
Recombinant Human CX3CL1 (Fractalkine) (carrier-free) BA
Recombinant Mouse CX3CL1 (Fractalkine) (carrier-free) BA
Recombinant Human CCL27 (CTACK) (carrier-free) BA
Recombinant Human CCL22 (MDC) (carrier-free) BA
FITC Annexin V Apoptosis Detection Kit with PI FC
Recombinant Mouse CCL24 (Eotaxin-2) (carrier-free) BA
Recombinant Human CCL24 (Eotaxin-2) (carrier-free) BA
Recombinant Human VEGF-165 (carrier-free) BA
Recombinant Human CCL28 (MEC) (carrier-free) BA
Recombinant Mouse CCL28 (MEC) (carrier-free) BA
Recombinant Mouse CCL1 (I-309) (carrier-free) BA
LEGEND MAX™ Human Myeloperoxidase ELISA Kit ELISA
Recombinant Human CCL20 (MIP-3α) (carrier-free) BA
FITC Annexin V Apoptosis Detection Kit with 7-AAD FC
Recombinant Mouse IL-33 (ELISA Std.) ELISA
Recombinant Human IL-22 (ELISA Std.) ELISA
Recombinant Human CXCL17 (VCC-1) (carrier-free) BA
Recombinant Mouse CXCL17 (VCC-1) (carrier-free) BA
Recombinant Human EGF (carrier-free) BA
Recombinant Mouse EGF (carrier-free) BA
Recombinant Mouse IL-6 (Animal-Free) BA
Recombinant Human TGF-β3 (carrier-free) BA
Recombinant Human IL-11 (carrier-free) BA
Recombinant Mouse CCL7 (MCP-3) (carrier-free) BA
Recombinant Mouse CCL9 (MIP-1γ) (carrier-free) BA
Recombinant Mouse CCL21 (6CKine) (carrier-free) BA
Recombinant Mouse Thrombopoietin (TPO) (Animal-Free) BA
Recombinant Mouse IL-3 (Animal-Free) BA
Recombinant Mouse IL-4 (Animal-Free) BA
Recombinant Human IL-21 (carrier-free) BA
Recombinant Mouse CXCL13 (carrier-free) BA
LEGEND MAX™ Human sICAM-1/CD54 ELISA Kit with Pre-coated Plates ELISA
Stop Solution for TMB Substrate ELISA
Zombie Aqua™ Fixable Viability Kit FC, ICFC, ICC
LEGEND MAX™ Human CXCL10 (IP-10) ELISA Kit ELISA
Recombinant Mouse CXCL7 (NAP-2) (carrier-free) BA
Recombinant Mouse CXCL3 (carrier-free) BA
Recombinant Mouse CCL25 (TECK) (carrier-free) BA
Recombinant Human CCL25 (TECK) (carrier-free) BA
Recombinant Human CCL23 (MPIF-1) (Arg46-Asn120) (carrier-free) BA
Recombinant Human Erythropoietin (EPO) (carrier-free) BA
Recombinant Mouse IL-25 (IL-17E) (insect expressed, carrier-free) BA
Recombinant Human FASL (TNFSF6) (carrier-free) BA
ELISA MAX™ Deluxe Set Human CXCL10 (IP-10) ELISA
Recombinant Mouse CXCL14 (BRAK) (carrier-free) BA
Recombinant Human CXCL14 (BRAK) (carrier-free) BA
Recombinant Mouse Erythropoietin (EPO) (carrier-free) BA
Recombinant Human CXCL12 (SDF-1β) (carrier free) BA
Recombinant Mouse CCL19 (MIP-3β) (carrier-free) BA
Recombinant Mouse CCL12 (MCP-5) (carrier-free) BA
Zombie NIR™ Fixable Viability Kit FC, ICFC
Recombinant Human VEGF-C (carrier-free) BA
Recombinant Mouse NGAL (Lipocalin-2) (carrier-free) BA
Recombinant Human NGAL (Lipocalin-2) (carrier-free) BA
Recombinant Mouse IGF-II (carrier-free) BA
Purified Annexin V FC
Recombinant Mouse CXCL12 (SDF-1β) (carrier-free) BA
Recombinant Human TGF-α (carrier-free) BA
Recombinant Rat CCL20 (MIP-3α) (carrier-free) BA
LEGEND MAX™ Human CCL17 (TARC) ELISA Kit ELISA
LEGEND MAX™ Human CCL5 (RANTES) ELISA Kit ELISA
Recombinant Mouse CXCL4 (PF-4) (carrier-free) BA
Accutase® Cell Detachment Solution FC
Recombinant Human IGF-II (carrier-free) BA
Recombinant Human IGF-I (carrier-free) BA
Recombinant Human TRANCE (RANKL) (carrier-free) BA
Recombinant Mouse BAFF (carrier-free) BA
Recombinant Human APRIL (TNFSF13) (ELISA Std.) ELISA
Recombinant Human IL-15 (ELISA Std.) ELISA
Recombinant Mouse IGF-I (carrier-free) BA
Recombinant Human sTNF-RII (TNFRSF1B) (carrier-free) BA
ELISA MAX™ Deluxe Set Human CCL17 (TARC) ELISA
Recombinant Mouse CTLA-4-Fc Chimera (carrier-free) BA
Recombinant Human CD40L (TNFSF5) (carrier-free) BA
Recombinant Human CTLA-4-Fc Chimera (carrier-free) BA
Recombinant Mouse TNF-α (Animal-Free) BA
Recombinant Human Activin A (carrier-free) BA
Recombinant Human FGF-4 (carrier-free) BA
Recombinant Human IL-18 (carrier-free) BA
Recombinant Rat GM-CSF (carrier-free) BA
Recombinant Human IFN-α2 (carrier-free) BA
ELISA MAX™ Deluxe Set Human CCL5 (RANTES) ELISA
ELISA MAX™ Deluxe Set Human CXCL5 ELISA
LEGEND MAX™ Mouse IL-35 Heterodimer ELISA Kit ELISA
ELISA MAX™ Deluxe Set Human CCL28 ELISA
Recombinant Rat Erythropoietin (EPO) (carrier-free) BA
Recombinant Human PCSK9 (carrier-free) BA
Recombinant Human CCL17 (TARC) (ELISA Std.) ELISA
Recombinant Human TIMP-1 (carrier-free) BA
Recombinant Human Arginase I (carrier-free) BA
Recombinant Human MMP-1 (carrier-free) BA
Recombinant Human CD14 (carrier-free) BA
Recombinant Human CTLA-4 (ELISA Std.) ELISA
Recombinant Mouse IL-34 (ELISA Std.) ELISA
Recombinant Human IL-34 (ELISA Std.) ELISA
Recombinant Human Latent TGF-β (ELISA Std.) ELISA
Recombinant Human Mesothelin-Fc Chimera (carrier-free) BA
Recombinant Mouse Thrombopoietin (TPO) (carrier-free) BA
Recombinant Human TIMP-2 (carrier-free) BA
Recombinant Mouse TIMP-1 (carrier-free) BA
Brilliant Violet 421™ Annexin V FC
Alexa Fluor® 488 Streptavidin FC, ICFC
Alexa Fluor® 647 Streptavidin FC, ICFC
Recombinant Mouse CCL3 (MIP-1α) (carrier-free) BA
Recombinant Human LIF (carrier-free) BA
Recombinant Mouse Mesothelin (carrier-free) BA
Recombinant Rat IFN-γ (ELISA Std.) ELISA
Recombinant Mouse IL-17A/F Heterodimer (ELISA Std.) ELISA
Recombinant Human TGF-β1 (ELISA Std.) ELISA
Zombie Violet™ Fixable Viability Kit FC, ICFC, ICC
Zombie Green™ Fixable Viability Kit FC, ICFC, ICC
Zombie UV™ Fixable Viability Kit FC, ICFC
CFSE Cell Division Tracker Kit ICFC, in-vivo Cell Tracking
LEGEND MAX™ Human Adiponectin (Acrp30) ELISA Kit ELISA
Bromodeoxyuridine (BrdU) FC, ICFC
Recombinant Mouse CCL5 (RANTES) (carrier-free) BA
Recombinant Mouse IFN-β (ELISA Std.) ELISA
Recombinant Human IL-9 (carrier-free) BA
Recombinant Mouse Latent TGF-β (ELISA Std.) ELISA
Recombinant Human MMP-3 (carrier-free) BA
APC Streptavidin (High Concentration) FC, ICFC
Recombinant Human CCL2 (MCP-1) (Animal-Free) BA
Recombinant Human VEGFR2-Fc Chimera (carrier-free) BA
Recombinant Human NT-4 (Animal-Free) BA
Cell Activation Cocktail (without Brefeldin A) Activ, FC, ICFC
Recombinant Human RBP4 (carrier-free) BA
Recombinant Rat IFN-γ (carrier-free) BA
Recombinant Human ICOS-Fc Chimera (carrier-free) BA
Cell Activation Cocktail (with Brefeldin A) Activ, FC, ICFC
Recombinant Mouse ICOS-Fc Chimera (carrier-free) BA
Recombinant Mouse VEGF-164 (Animal-Free) BA
Recombinant Human NT-3 (carrier-free) BA
Recombinant Human NT-4 (carrier-free) BA
Recombinant Human FGF-1-acidic (carrier-free) BA
Nunc™ MaxiSorp™ ELISA Plates, Uncoated ELISA
Plate Sealers ELISA
DRAQ7™ FC, ICFC
Recombinant Human WISP-1 (carrier-free) BA
Recombinant Human WNT-7a (carrier-free) BA
Recombinant Human BMP-4 (carrier-free) BA
Go To Top Version: 6    Revision Date: 01/27/2021

For research use only. Not for diagnostic use. Not for resale. BioLegend will not be held responsible for patent infringement or other violations that may occur with the use of our products.

 

*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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