AAT Bioquest

Live or Dead™ Cell Viability Assay Kit *Red/Blue Dual Fluorescence*


Excitation (nm)
Emission (nm)
This Live or Dead™ Cell Viability Assay Kit uses two fluorescent indicators: Cellbrite™ Red (Ex/Em = 613/631 nm) for labeling viable cells and a cell-impermeable DNA-binding dye Nuclear Blue™ DCS1 (Ex/Em = 360/450 nm) for labeling dead cells with damaged membranes. Cells grown in black-wall plates can be stained and quantified in less than two hours. The assay is more robust and accurate than the other viability assays. It can be readily adapted for a wide variety of fluorescence platforms such as microplate assays, fluorescence microscopes, and flow cytometry. The kit provides all the essential components with an optimized assay protocol. It is suitable for both proliferating and non-proliferating cells (either suspension or adherent cells).


Fluorescence microscope

ExcitationCy5 filter (alive), DAPI filter (dead)
EmissionCy5 filter (alive), DAPI filter (dead)
Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Excitation610, 360 nm
Emission650, 450 nm
Cutoff630, 420 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode


Example protocol


Protocol Summary
  1. Prepare cells with test compounds
  2. Add dye-working solution
  3. Incubate at room temperature or 37°C for 30 minutes to 1 hour
  4. Monitor fluorescence intensity (bottom read mode) at Ex/Em = 610/650 nm (Cutoff = 630 nm, Red) and Ex/Em = 360/450 nm (Cutoff =420 nm, Blue) or fluorescence microscope with Cy5 channel (live) and DAPI channel (dead)
Important Note

Thaw all the kit components at room temperature before starting the experiment.


For guidelines on cell sample preparation, please visit:



Add 5 µL of 200X Cellbrite™ Red (Component A) and 5 µL of 200X Nuclear Blue™ DCS1 (Component C) into 1 mL of Assay Buffer (Component B) and mix well to make dye-working solution. This dye-working solution is stable for at least 1 hour at room temperature.

Note: As the optimal staining conditions may vary depending on different cell types, it’s recommended to determine the appropriate concentration of Component A and C individually.


  1. Prepare cells according to the standard protocol. Note: We treated HeLa cells with staurosporine (SS) for 4 hours at 37ºC to induce cell apoptosis. See Figure 1 for details.
  2. Replace growth medium with 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of dye-working solution.
  3. Incubate the dye-working solution plate at room temperature or 37°C for 30 minutes to 1 hour, protected from light.
  4. Wash cells with HHBS, PBS or buffer of your choice twice.
  5. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Assay Buffer (Component B) into the cells.
  6. Monitor the fluorescence signal under a fluorescence microscope with Texas Red or Cy5 filter for live cells, and DAPI filter for dead cells. The fluorescence intensity can also be analyzed with a fluorescence microplate reader (bottom read mode) at Ex/Em = 610/650 nm (Cutoff = 630 nm, Red) and Ex/Em = 360/450 nm (Cutoff =420 nm, Blue).


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Spectral properties

Excitation (nm)612
Emission (nm)630



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Functional evidence that the self-renewal gene NANOG regulates esophageal squamous cancer development
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Localized functional chemical stimulation of TE 671 cells cultured on nanoporous membrane by calcein and acetylcholine
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A vaccination and challenge model using calcein marked fish
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Novel fluorescence assay using calcein-AM for the determination of human erythrocyte viability and aging
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Journal: Cytometry A (2005): 78
Cytotoxic effects of 100 reference compounds on Hep G2 and HeLa cells and of 60 compounds on ECC-1 and CHO cells. I mechanistic assays on ROS, glutathione depletion and calcein uptake
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Comparison of the usefulness of the MTT, ATP, and calcein assays to predict the potency of cytotoxic agents in various human cancer cell lines
Authors: Mueller H, Kassack MU, Wiese M.
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In vitro assay of mineralized-tissue formation on titanium using fluorescent staining with calcein blue
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Journal: Biomaterials (2003): 3885
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