Heat-killed (mixed 1:1 with live) C57BL/6 mouse splenocytes were stained with Zombie R685™ and analyzed before fixation (blue) or after fixation (red). Cells alone, without Zombie R685™ staining, are indicated in black.
Heat-killed (mixed 1:1 with live) C57BL/6 mouse splenocytes were stained with Zombie R685™ and analyzed before fixation (blue) or after fixation (red). Cells alone, without Zombie R685™ staining, are indicated in black.
Heat-killed C57BL/6 mouse thymocytes were stained with Apotracker™ Tetra PE and Zombie™ R685 (left) or stained with Apotracker™ Tetra PE only (right).
Zombie R685™ is an amine reactive fluorescent dye that is non-permeant to live cells, but permeant to the cells with compromised membranes. Thus, it can be used to assess live vs. dead status of mammalian cells. Zombie R685™ is a polar water soluble dye, making it suitable for use in multi-color detection.
Zombie R685™ Fixable Viability Kit is composed of lyophilized Zombie R685™ dye and anhydrous DMSO. For reconstitution, pre-warm the kit to room temperature; add 100 µL of DMSO to one vial of Zombie R685™ dye and mix until fully dissolved. 100 tests = 1 vial of Zombie R685™ + DMSO, 500 tests = 5 vials of Zombie R685™ + DMSO.
Storage & Handling
Store kit at -20°C upon receipt. Do not open vials until needed. Once the DMSO is added to the Zombie R685™ dye, 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.
Each lot of this product is quality control tested by immunofluorescent staining with flow cytometric analysis. For flow cytometry, the suggested dilution is 1:100-1:1000 for 1-10 million cells. It is recommended that the reagent be titrated for optimal performance for each application, as optimal dosage varies with cell type.
Excitation Laser
Red Laser (633 nm)
Application Notes
Zombie R685™ dye is excited by the red laser (633 nm) and has fluorescence emission maximum at 685 nm. If using in a multi-color panel design, filter optimization may be required depending on other fluorophores used. Zombie R685™ has similar spectra to Spark NIR™ 685.
Standard Cell Staining Protocol:
Prior to reconstitution, spin down the vial of lyophilized reagent in a microcentrofuge to ensure the reagent is at the bottom of the vial.
For reconstitution, pre-warm the kit to room temperature; add 100 µL of DMSO to one vial of Zombie R685™ dye and mix until fully dissolved.
Wash cells with PBS buffer (no Tris buffer and protein free).
Dilute Zombie R685™ dye at 1:100-1000 in PBS. Resuspend 1-10 x 106 cells in diluted 100 µL Zombie R685™ 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.
Incubate the cells at room temperature, in the dark, for 15-30 minutes.
Wash one time with 2 mL BioLegend’s Cell Staining Buffer (Cat. No. 420201) or equivalent buffer containing serum or BSA.
Continue performing antibody staining procedure as desired.
Cells can be fixed with paraformaldehyde or methanol prior to permeabilization or can be analyzed without fixation.
No-wash Sequential Staining Protocol:
Wash cells with PBS buffer (no Tris buffer and protein free).
For reconstitution, pre-warm the kit to room temperature; add 100 µL of DMSO to one vial of Zombie R685™ dye and mix until fully dissolved.
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 R685™ 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 R685™ solution. Resuspend 1-10 x 106 cells in the appropriate volume of Zombie R685™ 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.
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.
Add 1-2 mL Cell Staining Buffer (Cat. No. 420201) or equivalent buffer containing BSA or serum. Centrifuge to pellet.
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 R685™ 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 R685™ may be required since the BSA/serum will react with and bind up some proportion of the Zombie R685™.
