For flow cytometry experiments, it is imperative to make sure samples are clean of debris and dying cells as they can non-specifically bind to dyes and antibodies meant to stain live cells. Dead cell exclusion is even more critical when working with small populations or rare events, as even small changes can have a big impact on your analysis. In addition, certain cell types (e.g., bone marrow resident plasma cells or mouse intestinal lamina propria cells) are more sensitive than others, and lengthy protocols for sample isolation can affect the viability of your samples. In these cases, it is particularly important to exclude dead cells from your analysis.

 

Chemical probes that target nucleic acids, such as 7-AAD or propidium iodide, are commonly used to differentiate live and dead cells. These dyes cannot penetrate the intact membranes of healthy cells but can access and stain nucleic acids through the disrupted membranes of dying cells. However, nucleic acid stains are not perfect for every occasion.

 

First, they cannot be used for viability staining on cells that will be fixed or permeabilized, as the live/dead staining pattern is not preserved through treatments that disrupt cell membranes. Second, nucleic acid probes can be limited in their excitation and emission properties, restricting options for their use in multicolor panel design.

 

In contrast, our amine-reactive Zombie Dyes are compatible with fixation and intracellular staining techniques and are offered in a wide range of colors – from our traditional offerings that can be used in the most common conventional flow cytometry channels to dyes designed to emit at unique wavelengths for large, optimized panel design, such as Zombie B550™, Zombie YG581™, Zombie R685™, and Zombie R718™.

 

Zombie Dye	Excitation Laser	Emission Max
Zombie UV™	UV 355 nm	459 nm
Zombie Violet™	Violet 405 nm	423 nm
Zombie Aqua™	Violet 405 nm	516 nm
Zombie Yellow™	Violet 405 nm	572 nm
Zombie Green™	Blue 488 nm	515 nm
Zombie B550™	Blue 488 nm	540 nm
Zombie YG581™	Yellow/Green 561 nm	581 nm
Zombie Red	Yellow/Green 561 nm	624 nm
Zombie R685™	Red 633 nm	685 nm
Zombie R718™	Red 633 nm	718 nm
Zombie NIR™	Red 633 nm	746 nm

 

Learn more about Zombie Dyes.

Mouse splenocytes were stored overnight to allow dead cells to accumulate. Cells were then stained with Zombie Dyes as indicated and analyzed before fixation (purple) or after fixation and permeabilization (red). Cells alone, without Zombie staining, are indicated in black.

 

Zombie Dyes detect amines on the surface of all cells at a low level but intensely stain dead and dying cells by accessing the abundant intracellular amines through disrupted membranes. As this covalent interaction is irreversible, cells can be washed, fixed, and permeabilized after viability staining without altering the initial live/dead staining pattern.

 

(Left) Impermeant nucleic acid stains can only stain DNA when the membrane is compromised. (Right) Zombie dyes bind to primary amines on proteins. Live cells receive low-level surface staining, while dead cells stain at a much brighter fluorescence level due to the exposure of internal proteins.

 

Whether you are interested in Zombie Dyes or any of our other chemical probes, you can always talk with our experts to determine which reagent is best for your experimental setup. To probe beyond live/dead discrimination, explore our growing portfolio of antibody and non-antibody based methods of assessing cell health and proliferation. With our products and guidance, we will ensure your data is consistent, trustworthy, and groundbreaking.