All New Zombie Dyes

"Don't look back."
The Walking Dead, AMC Network.
 
We've previously discussed why it's a good idea for you to incorporate a live/dead indicator dye in your panel for flow analysis here. And, BioLegend offers a line of Zombie dyes, which recognize primary amines. On live cells, primary amines on proteins are only labeled on the cell surface. As cells die, they lose their membrane integrity, which allows the Zombie dyes to access cytoplasmic proteins.  Due to the significantly higher number of proteins on the inside of the cell compared to the surface, Zombie staining of dead cells will be much higher than live cells.

Your apoptosing cells.
PI, an impermeant dye, and 7-AAD, a semi-permeant dye, are common nucleic acid stains often used as live/dead indicators in flow cytometry. However, they aren't perfect for every occasion. Since nucleic acid stains become fluorescent when bound to DNA, their staining pattern can be affected by any organic denaturing solvents, like methanol (MeOH) or ethanol (EtOH). Both MeOH and EtOH are often used in fixatives for cytokine detection, intranuclear antigen detection, or Phospho-flow.

These solvents can denature the DNA, causing the nucleic acid stain to lose affinity (as you see in the reduction of signal with 7-AAD) and wash away.
  MeOH/EtOH fixation can reduce 7-AAD binding and a loss of signal. Day-old unfixed spleen cells are on the left. The same cells were fixed with 70% EtOH, 1.5% PFA are on the right.  
Or in the case with PI, the solvents will permeabilize the previously live cells, in addition to causing the nucleic acid stain come off the DNA of dead cells. This results in PI very quickly labeling the newly permeabilized cells that had been alive prior to EtOH/MeOH fixation, causing a very big shift in "background", thus losing resolution of live and dead cells. 
  MeOH/EtOH fixation can unwind the DNA and cause PI to pop off and find new targets that were originally alive to begin with, causing an increase in background. Day-old unfixed spleen cells are on the left. The same cells were fixed with 70% EtOH, 1.5% PFA are on the right.  

Don't fall for false positives. WWE Entertainment.
As previously mentioned, the Zombie dyes covalently react with primary amines, which is an irreversible conjugation to the proteins of the cell. MeOH and EtOH fixation won't affect the Zombies, so you can trust your final results. And, we're proud to announce four new additions (UV, Violet, Green, and Red) to round out our Zombie line.

*Indicates new release.
Curious about testing our cost-effective Zombie dyes? Contact our sales team here. You can learn more about the Zombie dyes, watch our Zombie Apocalypse video, view protocols, learn about our promotion, and get more Zombie information on our page here.
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