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TWO-PRO™ 1 [equivalent to TO-PRO®-1] *5 mM DMSO Solution* *CAS 157199-59-2*

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Physical properties
Molecular weight645.38
Spectral properties
Extinction coefficient (cm -1 M -1)628001
Excitation (nm)515
Emission (nm)531
Quantum yield0.251
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™-1 is chemically equivalent to TO-PRO®-1 (TO-PRO® is the trademark of Invitrogen). TWO-PRO™-1 is a carbocyanine dimer with green fluorescence similar to FITC. It is cell-impermeant and easily distinguished from Cy5 and rhodamines 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™-1 gives strong and selective nuclear staining in cultured cells and in paraffin sections. Simultaneous labeling with cell-permeable Nuclear Red™ LCS1 dye and cell-impermeant TWO-PRO™-1 can be used to assess cell viability. TWO-PRO™-1 and Nuclear Red™ both have much greater extinction coefficients than that of DNA-bound propidium iodide.


Fluorescence microscope

ExcitationFITC filter set
EmissionFITC filter set
Recommended plateBlack wall/clear bottom
Instrument specification(s)FITC filter set

Example protocol


TWO-PRO™-1 working solution
Make TWO-PRO™-1 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 TWO-PRO™-1 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 FITC filter. 


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of TWO-PRO™ 1 [equivalent to TO-PRO®-1] *5 mM DMSO Solution* *CAS 157199-59-2* 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 mM154.947 µL774.737 µL1.549 mL7.747 mL15.495 mL
5 mM30.989 µL154.947 µL309.895 µL1.549 mL3.099 mL
10 mM15.495 µL77.474 µL154.947 µL774.737 µL1.549 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)628001
Excitation (nm)515
Emission (nm)531
Quantum yield0.251

Product Family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yield
TWO-PRO™ 3 [equivalent to TO-PRO®-3] *5 mM DMSO Solution* *CAS 157199-63-8*64265710200010.111



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A toolkit for the identification of NEAT1\_2/paraspeckle modulators
Authors: An, Haiyan and Elvers, Karen T and Gillespie, Jason A and Jones, Kimberley and Atack, John R and Grubisha, Olivera and Shelkovnikova, Tatyana A
Journal: Nucleic Acids Research (2022): e119--e119


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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
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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.
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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
SOCS-3 antagonizes pro-apoptotic effects of TRAIL and resveratrol in prostate cancer cells
Authors: Horndasch M, Culig Z.
Journal: Prostate (2011): 1357
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
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)