Zombie Dye Staining Protocol

Reagent List

• PBS (without sodium azide, Tris, or any additional protein)
• Zombie Fixable Viability Kit
• Cell Staining Buffer (BioLegend Cat. No. 420201)
• Fixation Buffer (Cat. No. 420802) or equivalent

Reconstitute Zombie Dye:
 

1. Spin down the vial of lyophilized reagent in a microcentrifuge to ensure the reagent is at the bottom of the vial.
    
2. Pre-warm the kit to room temperature; add 100 µL of DMSO to one vial of Zombie dye and mix until fully dissolved.
    

• Use immediately, or store at -20°C in a dry place and protected from light, preferably in a desiccator or in a container with desiccant for no more than one month.

 

Standard Cell Staining Protocol:

 

1. Wash cells with PBS (sodium azide, Tris, and protein free).

 

2. Dilute Zombie Dye in PBS that does not contain any BSA, serum, or other soluble proteins at the optimized concentration for your cell type. We recommend a 1:100-1:1000 dilution range, but optimal dosage will vary by application. Titrate the amount of dye and/or number of cells per 100 µL for optimal performance.

 

3. Resuspend 1-10 x 10⁶ cells in 100 µL of diluted Zombie solution.

 

4. Incubate the cells at room temperature, in the dark, for 15-30 minutes.

 

5. Add 2 mL of Cell Staining Buffer or equivalent buffer containing BSA or serum. Centrifuge to pellet and decant.

 

6. Cells can be fixed with paraformaldehyde or methanol prior to permeabilization or can be analyzed without fixation.

 

7. Continue antibody staining and analysis as desired.

 

No-wash Sequential Staining Protocol:

 

1. Dilute Zombie Dye into PBS that does not contain any BSA, serum, or other soluble proteins at the optimized concentration for your cell type. We recommend a 1:100-1:1000 dilution range, but optimal dosage will vary with cell type.

 

2. Resuspend 1-10 million cells per tube in diluted Zombie solution. The final volume of Zombie Dye/PBS should be 100 µL minus the volume of antibody that will be added after this step is complete. For example, if you are adding 20 µL of antibody cocktail, use 80 µL of Zombie Dye solution.

 

3. Incubate for 10-15 minutes at room temperature, protected from light. Without washing the cells, add the cell surface antibodies and incubate for another 15-20 min.

 

4. Add 1-2 mL Cell Staining Buffer or equivalent buffer containing BSA or serum. Centrifuge to pellet.

 

5. Continue with normal fixation and permeabilization procedure. If skipping fixation step to analyze live cells, complete an additional wash step to minimize background staining 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 absorb some of the Zombie Dye.