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.