![]() ![]() However, we would not recommend using BV510™ off the violet laser and Zombie Aqua™ off the UV laser at the same time. Q: Can I use the UV laser to stimulate Zombie Aqua™? If so, can I then use it in conjunction with BV510™?Ī: While we typically do not test Zombie Aqua™ with the UV laser, its excitation peak suggests it is effectively excited at 355 nm. 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: How does the performance of your Zombie dyes compare with competitors?Ī: Zombie dyes have been tested against other leading competitors’ fixable viability kits and given comparable results. Dead cells free#Light exposure or reagents with hydrogen peroxide can lead to free radical formation, affecting fluorescence. However, it should be noted that Zombie dyes can still be sensitive to reactive oxygen species. Q: Can I use methanol/ethanol for fixation after using Zombie dyes?Ī: Yes, most fixation reagents are fine to be used with Zombie dyes. 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: Why can’t I fix my cells prior to using Zombie dyes?Ī: The fixation process can contort and alter the membrane of cells, effectively rendering them as dead. 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. 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. If planning to skip fixation and analyze cells live, complete an additional wash step to minimize any unnecessary background of the live cells. Continue with normal fixation and permeabilization procedure. 420201) or equivalent buffer containing BSA or serum. Without washing the cells, add the cell surface antibodies and incubate for another 15-20 min.Ĥ. Incubate for 10-15 minutes at RT, protected from light. For example, if you are adding 20 µl of antibody cocktail, use 80 µl of Zombie dye solution.ģ. Final volume of Zombie dye/PBS should be 100 µl minus the volume of antibody that will be added after this step is complete. ![]() Resuspend 1-10 million cells per tube in the prepared Zombie dye/PBS solution. We recommend a 1:100-1:1000 dilution range, but optimal dosage will vary with cell type.Ģ. Dilute Zombie dye into PBS that does not contain any BSA, serum, or other soluble proteins at the optimized concentration for your cell type. Continue performing antibody staining procedure as desired.ġ. Cells can be fixed with paraformaldehyde or methanol prior to permeabilization or can be analyzed without fixation.ħ. 420201) or equivalent buffer containing serum or BSA.Ħ. Wash one time with 2 ml BioLegend’s Cell Staining Buffer (Cat. Incubate the cells at room temperature, in the dark, for 15-30 minutes.ĥ. However, 1 µl of reagent has been found to be good for most types of cells.Ĥ. Note: The amount of dye used can also influence the ability to detect apoptotic as well as live and dead cells. Different cell types can have a wide degree of variability in staining. To minimize background staining of live cells, titrate the dye for optimal performance. Add 1 µl of Zombie dye to 100 µl of cells. Note: Be sure that the PBS does not contain sodium azide, Tris, or any other protein.ģ. Resuspend cells at 1.0 x 106 cells/100 µl of PBS. Wash cells with PBS (sodium azide, Tris, and protein free).Ģ. ![]()
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