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DiTO™-3 [equivalent to TOTO®-3] *5 mM DMSO Solution*

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Physical properties
Molecular weight1354.85
Spectral properties
Extinction coefficient (cm -1 M -1)1540001
Excitation (nm)642
Emission (nm)661
Quantum yield0.061
Storage, safety and handling
H-phraseH303, H313, H340
Hazard symbolT
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R68
StorageFreeze (< -15 °C); Minimize light exposure


Molecular weight
Extinction coefficient (cm -1 M -1)
Excitation (nm)
Emission (nm)
Quantum yield
DiTO™-3 is chemically equivalent to TOTO®-3 (TOTO® is the trademark of Invitrogen). DiTO™-3 is a carbocyanine dimer with far-red fluorescence similar to Cy® 5 dyes. It is cell-impermeant and easily distinguished from fluorescein and rhodamine as a nuclear counterstain and dead cell indicator. It is non-fluorescent in the absence of nucleic acids, and generates a very bright fluorescence signal upon binding to DNA. DiTO™-3 gives strong and selective nuclear staining in cultured cells and in paraffin sections. Simultaneous labeling with cell-permeable Nuclear Green™ LCS1 dye and cell-impermeant DiTO®-3 can be used to assess cell viability. DiTO™-3 and Nuclear Green™ both have much greater extinction coefficients than that of DNA-bound propidium iodide.


Fluorescence microscope

ExcitationCy5 filter set
EmissionCy5 filter set
Recommended plateBlack wall/clear bottom
Instrument specification(s)Cy5 filter set

Example protocol


DiTO™-3 working solution
Make DiTO™-3 working solution in Hanks with 20 mM Hepes buffer (HH buffer) or buffer of your choice at your desired concentration.
Note     In initial experiments, it may be best to try several dye concentrations to determine the optimal concentration that yields the desired result. High dye concentration tends to cause nonspecific staining of other cellular structures.


Caution: The following protocol can be adapted for most cell types. Growth medium, cell density, the presence of other cell types and factors may influence staining. Residual detergent on glassware may also affect staining of many organisms, and cause brightly stained material to appear in solutions with or without cells present.
  1. Grow and treat cells as desired.
  2. Remove the cell culture medium and fix cells.
  3. Add DiTO™-3 working solution (1 to 10 µM) into the cells (either suspension or adherent cells), and stain the cells for 15 to 60 minutes.
  4. Remove the dye working solution and add HH buffer or buffer of your choice.
  5. Analyze the cellular staining with a fluorescence microscope using Cy5 filter. 


Common stock solution preparation

Table 1. Volume of Water needed to reconstitute specific mass of DiTO™-3 [equivalent to TOTO®-3] *5 mM DMSO Solution* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM73.809 µL369.045 µL738.089 µL3.69 mL7.381 mL
5 mM14.762 µL73.809 µL147.618 µL738.089 µL1.476 mL
10 mM7.381 µL36.904 µL73.809 µL369.045 µL738.089 µL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles


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Spectral properties

Extinction coefficient (cm -1 M -1)1540001
Excitation (nm)642
Emission (nm)661
Quantum yield0.061



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Chromatin staining of Drosophila testes
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Journal: Cold Spring Harb Protoc (2012)
In vivo cancer imaging by poly(ethylene glycol)-b-poly(varepsilon-caprolactone) micelles containing a near-infrared probe
Authors: Cho H, Indig GL, Weichert J, Shin HC, Kwon GS.
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The fluorescent dyes TO-PRO-3 and TOTO-3 iodide allow detection of microbial cells in soil samples without interference from background fluorescence
Authors: Henneberger R, Birch D, Bergquist P, Walter M, Anitori RP.
Journal: Biotechniques (2011): 190
Effect of Mg ions on efficiency of gene electrotransfer and on cell electropermeabilization
Authors: Haberl S, Miklavcic D, Pavlin M.
Journal: Bioelectrochemistry (2010): 265
Visualizing nuclei in skin cryosections: viable options to 4'6-diamidino-2-phenylindol for confocal laser microscopy
Authors: Glaser K, Wilke K, Wepf R, Biel SS.
Journal: Skin Res Technol (2008): 324
Highly chlorinated Escherichia coli cannot be stained by propidium iodide
Authors: Phe MH, Dossot M, Guilloteau H, Block JC.
Journal: Can J Microbiol (2007): 664
DNA labeling in living cells
Authors: Martin RM, Leonhardt H, Cardoso MC.
Journal: Cytometry A (2005): 45
Quinolinium, 1,1'-[1,3-propanediylbis[(dimethyliminio)-3,1-propanediyl]]bis[4-[3-(3-methyl-2(3 H)-benzothiazolylidene)-1-propen-1-yl]-,iodide (1:4)
Authors: Shan L., undefined
Journal: In: Molecular Imaging and Contrast Agent Database (MICAD), Bethesda (MD). (2004)
Chlorination effect on the fluorescence of nucleic acid staining dyes
Authors: Phe MH, Dossot M, Block JC.
Journal: Water Res (2004): 3729
Sensitive and reliable JC-1 and TOTO-3 double staining to assess mitochondrial transmembrane potential and plasma membrane integrity: interest for cell death investigations
Authors: Zuliani T, Duval R, Jayat C, Schnebert S, Andre P, Dumas M, Ratinaud MH.
Journal: Cytometry A (2003): 100