It is critical to understand the degree of cell death in any flow cytometry assay and exclude those cells from the analysis. BioLegend provides DNA dyes, Helix NP™ NIRDRAQ7™Propidium Iodide and 7-AAD, that enter and stain dead cells, but are impermeable to live cells for rapid, cost-effective analysis of unfixed cells. In cases where cell fixation is required, we now introduce fixable Zombie Aqua™Zombie Green™Zombie NIR™Zombie Red™Zombie Violet™Zombie UV™, and Zombie Yellow™. Learn more about our tools for live/dead cell discrimination below…

 

Not sure which Zombie is right for you? Try our Zombie Fixable Viability™ Sampler Kit, which contains 100 tests each of the UV, NIR, Violet, Aqua, and Yellow variants. 

Zombie Dyes

Zombie Dyes

*DRAQ7™ is a trademark of Biostatus Limited.

Zombie Dyes

  • Zombie Aqua is excited by both the violet laser (405nm) and the UV laser (350 nm), and it emits maximally at 516 nm
  • Zombie Green is excited by the blue laser (488nm) and emits maximally at 515 nm
  • Zombie NIT is excited by the red laser (633nm) and emits maximally at 746 nm
  • Zombie Red is excited by the yellow/green laser (561nm) and emits maximally at 624 nm  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.
  • Zombie UV is excited by the UV laser (350 nm) and emits maximally at 459 nm
  • Zombie Violet is excited by the violet laser (405nm) and emits maximally at 423 nm. Zombie Violet has similar emission to Brilliant Violet 421 and Pacific Blue.
  • Zombie Yellow is excited by the violet laser (405nm) and partially excited by the UV laser (350nm), and emits maximally at 572 nm.  Zombie Yellow has similar emission to Brilliant Violet 570.

 

Name Size Catalog No.
Zombie Aqua™ Fixable Viability Kit 100 tests/500 tests 423101/423102
Zombie Green™ Fixable Viability Kit 100 tests/500 tests 423111/423212
Zombie NIR™ Fixable Viability Kit 100 tests/500 tests 423105/423106
Zombie Red™ Fixable Viability Kit 100 tests/500 tests 423109/423110
Zombie UV™ Fixable Viability Kit 100 tests/500 tests 423107/423108
Zombie Violet™ Fixable Viability Kit 100 tests/500 tests 423113/423114
Zombie Yellow™ Fixable Viability Kit 100 tests/500 tests 423103/423104
Zombie Fixable Viability™ Sampler Kit 1 kit (500 tests total) 423117

Standard Cell Staining Protocol: 
1. Wash cells with PBS (sodium azide, Tris, and protein free). 
2. Resuspend cells at 1.0 x 106 cells/100 µl of PBS. 
Note: Be sure that the PBS does not contain sodium azide, Tris, or any other protein.
3. Add 1 µl of Zombie dye to 100 µl of cells.  To minimize background staining of live cells, titrate the dye for optimal performance.  Different cell types can have a wide degree of variability in staining. 
Note: The amount of dye used can also influence the ability to detect apoptotic as well as live and dead cells. However, 1 µl of reagent has been found to be good for most types of cells. 
4. Incubate the cells at room temperature, in the dark, for 15-30 minutes. 
5. Wash one time with 2 ml BioLegend’s Cell Staining Buffer (Cat. No. 420201) or equivalent buffer containing serum or BSA. 
6. Cells can be fixed with paraformaldehyde or methanol prior to permeabilization or can be analyzed without fixation. 
7. Continue performing antibody staining procedure as desired.

No-wash Sequential Staining Protocol:
1. Dilute Zombie dye into PBS that does not contain any BSA, serum, or other soluble proteins at the optimized concentration for your cell type. We recommend a 1:100-1:1000 dilution range, but optimal dosage will vary with cell type. 
2. Resuspend 1-10 million cells per tube in the prepared Zombie dye/PBS solution. Final volume of Zombie dye/PBS should be 100 µl minus the volume of antibody that will be added after this step is complete. For example, if you are adding 20 µl of antibody cocktail, use 80 µl of Zombie dye solution. 
3. Incubate for 10-15 minutes at RT, protected from light. Without washing the cells, add the cell surface antibodies and incubate for another 15-20 min. 
4. Add 1-2 mL Cell Staining Buffer (Cat. No. 420201) or equivalent buffer containing BSA or serum. Centrifuge to pellet.
5. 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. 

FAQs

 

 

Q: Why can’t I fix my cells prior to using Zombie dyes?
A: The fixation process can contort and alter the membrane of cells, effectively rendering them as dead. Since the ability of Zombie Aqua™, Zombie NIR™ and Zombie Yellow™ to stain dead cells is correlated with cell permeability, your results may no longer be a valid representation of dead versus live cells.

 

 

Q: Can I use methanol/ethanol for fixation after using Zombie dyes?
A: 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.

 

Q: How does the performance of your Zombie dyes compare with competitors?
A: 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.

 

Q: Can I use the UV laser to stimulate Zombie Aqua™? If so, can I then use it in conjunction with BV510™?
A: While we typically do not test Zombie Aqua™ with the UV laser, its excitation peak suggests it is effectively excited at 355 nm. However, we would not recommend using BV510™ off the violet laser and Zombie Aqua™ off the UV laser at the same time. Due to cross-beam excitation of BV510™ by the UV laser and the violet excitation of Zombie Aqua™, this would lead to significantly increased background and excessive compensation requirements.

 

 

Q: Can I use Zombie dyes to detect apoptotic cells?
A: Yes, Zombie dyes 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 dyes over PI and 7-AAD is that you can now fix and/or permeabilize the cells to stain for cell surface and intracellular antigens.

 

 

Q: Is Zombie Aqua™ an equivalent to cell labeling and tracer dyes such as CellTrace™ Violet and CFSE?
A: Zombie Aqua™ is not a direct equivalent to such dyes because their mechanism of action and scope of application are entirely different.


Q: I am concerned about the spillover I am observing from the Zombie dye into its neighboring channels.
A: The 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.


Q: Can Zombie be used to determine bacteria, yeast viability?
A: 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.


Q: Can I use Zombie with cells suspension containing serum?
A: 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.


Q: Can I use your Zombie dyes for microscopy application?
A: 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.

 

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