Zombie Fixable Viability™ Sampler Kit

Each lot of this reagent is quality control tested by immunofluorescent staining with flow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is 1:100-1:1000 dilution per 1-10 million cells in 100 µL volume. For immunocytochemistry, the suggested dilution is 1:1000. It is recommended that the reagent be titrated for optimal performance for each application, as optimal dosage varies with cell type.

Standard Cell Staining Protocol:

1. Prior to reconstitution, spin down the vial of lyophilized reagent in a microcentrofuge to ensure the reagent is at the bottom of the vial.
2. For reconstitution, pre-warm the kit to room temperature; add 100 µl of DMSO to one vial of Zombie dye and mix until fully dissolved
3. Wash cells with PBS buffer (no Tris buffer and protein free).
4. Dilute Zombie dye at 1:100-1000 in PBS. Resuspend 1-10 x 10⁶ cells in diluted 100 µl Zombie solution. To minimize background staining of live cells, titrate the amount of dye and/or number of cells per 100 µl for optimal performance. Different cell types can have a wide degree of variability in staining based on cell size and degree of cell death.
   
   Note: Don’t use Tris buffer as a diluent and be sure that the PBS does not contain any other protein like BSA or FBS.
   Note: The amount of dye used can also influence the ability to detect apoptotic as well as live and dead cells.


5. Incubate the cells at room temperature, in the dark, for 15-30 minutes.
6. Wash one time with 2 ml BioLegend’s Cell Staining Buffer (Cat. No. 420201) or equivalent buffer containing serum or BSA.
7. Continue performing antibody staining procedure as desired.
8. Cells can be fixed with paraformaldehyde or methanol prior to permeabilization or can be analyzed without fixation.

No-wash Sequential Staining Protocol:

1. Wash cells with PBS buffer (no Tris buffer and protein free).
2. For reconstitution, pre-warm the kit to room temperature; add 100 µl of DMSO to one vial of Zombie dye and mix until fully  dissolved
3. Determine the total µl volume of antibody cocktail previously titrated and optimized for the assay that will be added to each vial/well of cells based on a final volume of 100 µl. Subtract that antibody volume from the 100 µl total staining volume intended for the assay. In the remaining volume, dilute Zombie dye at 1:100-1000 in PBS as determined by prior optimization at that volume. For example, if you are adding 20 µl of antibody cocktail for a 100 µl total staining volume, use 80 µl of Zombie solution. Resuspend 1-10 x 10⁶ cells in the appropriate volume of Zombie solution. Different cell types can have a wide degree of variability in staining based on cell size and degree of cell death.
   
   Note: Don’t use Tris buffer as a diluent and be sure that the PBS does not contain any other protein like BSA or FBS.
   Note: The amount of dye used can also influence the ability to detect apoptotic as well as live and dead cells.


4. Incubate for 10-15 minutes at RT, protected from light. Without washing the cells, add the cell surface antibody cocktail and incubate for another 15-20 minutes.
5. Add 1-2 mL Cell Staining Buffer (Cat. No. 420201) or equivalent buffer containing BSA or serum. Centrifuge to pellet.
6. Continue with normal fixation and permeabilization procedure. If planning to skip fixation and analyze cells live, complete an additional wash step to minimize any unnecessary background of the live cells.

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. 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.

1. Lin J, et al. 2022. Adv Sci (Weinh). 9:e2202633. PubMed
2. Nigam N, et al. 2023. Cell Rep. 112823:42. PubMed