Live/Dead Controls...

Are most appropriate when you want to exclude dead cells and debris from your analysis.

Since dead cells and debris can non-specifically stain with antibodies and can also have antigen expression that is not consistent with live cells, it is helpful to exclude them from analysis. DNA stains like Propidium IodideHelix NP™ NIR, and Helix NP™ Green are cell membrane impermeant, meaning they only enter cells with compromised membranes, such as dead cells. For cells that will otherwise be analyzed unfixed, nucleic acid/DNA stains are the preferred live/dead indicator.

For samples that need to be fixed and permeabilized (particularly with ethanol or methanol), DNA binding dyes are often not ideal. This is because the DNA can be denatured, thereby releasing intercalated dyes and allowing them to non-specifically bind cells that are now fixed, permeabilized and thus “dead”. In addition, cells that were originally dead may lose their signal amplitude as the dyes escape. For these experiments, we highly recommend Zombie dyes, which covalently attach to primary amines instead of DNA. If the cell is alive when labeled, only amines at the cell surface will be bound. If the cells are dead when labeled, the amplitude of the signal will be several folds higher since the dye now has access to intracellular, amine-containing proteins. Zombie dyes are helpful for studies involving cytokine and transcription factor analysis requiring fixation and permeabilization.

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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.