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TWO-PRO™ 3 [equivalent to TO-PRO®-3] *5 mM DMSO Solution* *CAS 157199-63-8*

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Physical properties
Molecular weight671.42
Spectral properties
Extinction coefficient (cm -1 M -1)1020001
Excitation (nm)642
Emission (nm)657
Quantum yield0.111
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
TWO-PRO™-3 is chemically equivalent to TO-PRO®-3 (TO-PRO® is the trademark of Invitrogen). TWO-PRO™-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. TWO-PRO™-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 TWO-PRO™-3 can be used to assess cell viability. TWO-PRO™-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


TWO-PRO™-3 working solution
Make TWO-PRO™-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.
  3. Add TWO-PRO™-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 DMSO needed to reconstitute specific mass of TWO-PRO™ 3 [equivalent to TO-PRO®-3] *5 mM DMSO Solution* *CAS 157199-63-8* 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 mM148.938 µL744.69 µL1.489 mL7.447 mL14.894 mL
5 mM29.788 µL148.938 µL297.876 µL1.489 mL2.979 mL
10 mM14.894 µL74.469 µL148.938 µL744.69 µL1.489 mL

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)1020001
Excitation (nm)642
Emission (nm)657
Quantum yield0.111

Product Family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yield
TWO-PRO™ 1 [equivalent to TO-PRO®-1] *5 mM DMSO Solution* *CAS 157199-59-2*5155316280010.251
PRO-TAO-3 [equivalent to TO-PRO-3]6426571020000.111



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Journal: Pharmaceutics (2024): 70
A lysosomal regulatory circuit essential for the development and function of microglia
Authors: Iyer, Harini and Shen, Kimberle and Meireles, Ana M and Talbot, William S
Journal: Science advances (2022): eabp8321


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Daptomycin exerts rapid bactericidal activity against Bacillus anthracis without disrupting membrane integrity
Authors: Xing YH, Wang W, Dai SQ, Liu TY, Tan JJ, Qu GL, Li YX, Ling Y, Liu G, Fu XQ, Chen HP.
Journal: Acta Pharmacol Sin (2014): 211
Quantification of Candida albicans by flow cytometry using TO-PRO((R))-3 iodide as a single-stain viability dye
Authors: Kerstens M, Boulet G, Pintelon I, Hellings M, Voeten L, Delputte P, Maes L, Cos P.
Journal: J Microbiol Methods (2013): 189
A silicon cell cycle in a bacterial model of calcium phosphate mineralogenesis
Authors: Linton KM, Tapping CR, Adams DG, Carter RD, Shore RC, Aaron JE.
Journal: Micron (2013): 419
Pulsed electromagnetic field affects intrinsic and endoplasmatic reticulum apoptosis induction pathways in MonoMac6 cell line culture
Authors: Kaszuba-Zwoinska J, Chorobik P, Juszczak K, Zaraska W, Thor PJ.
Journal: J Physiol Pharmacol (2012): 537
Determination of the drug-DNA binding modes using fluorescence-based assays
Authors: Williams AK, Dasilva SC, Bhatta A, Rawal B, Liu M, Korobkova EA.
Journal: Anal Biochem (2012): 66
Transient changes in neuronal cell membrane permeability after blast exposure
Authors: Arun P, Abu-Taleb R, Valiyaveettil M, Wang Y, Long JB, Nambiar MP.
Journal: Neuroreport (2012): 342
A comparison of TO-PRO-1 iodide and 5-CFDA-AM staining methods for assessing viability of planktonic algae with epifluorescence microscopy
Authors: Gorokhova E, Mattsson L, Sundstrom AM.
Journal: J Microbiol Methods (2012): 216
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
SOCS-3 antagonizes pro-apoptotic effects of TRAIL and resveratrol in prostate cancer cells
Authors: Horndasch M, Culig Z.
Journal: Prostate (2011): 1357
Preclinical evaluation of novel triphenylphosphonium salts with broad-spectrum activity
Authors: Millard M, Pathania D, Shabaik Y, Taheri L, Deng J, Neamati N.
Journal: PLoS One (2010)