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

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Physical properties

Molecular weight	671.42
Solvent	DMSO

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	102000¹
Excitation (nm)	642
Emission (nm)	657
Quantum yield	0.11¹

Storage, safety and handling

H-phrase	H303, H313, H340
Hazard symbol	T
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R68
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	41116134

Overview SDS Protocol

CAS

157199-63-8

Molecular weight

671.42

Extinction coefficient (cm ^-1 M ^-1)

102000¹

Excitation (nm)

642

Emission (nm)

657

Quantum yield

0.11¹

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.

Platform

Fluorescence microscope

Excitation	Cy5 filter set
Emission	Cy5 filter set
Recommended plate	Black wall/clear bottom
Instrument specification(s)	Cy5 filter set

Example protocol

PREPARATION OF WORKING SOLUTION

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.

SAMPLE EXPERIMENTAL PROTOCOL

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.

Grow and treat cells as desired.
Remove the cell culture medium.
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.
Remove the dye working solution and add HH buffer or buffer of your choice.
Analyze the cellular staining with a fluorescence microscope using Cy5 filter.

Calculators

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 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	148.938 µL	744.69 µL	1.489 mL	7.447 mL	14.894 mL
5 mM	29.788 µL	148.938 µL	297.876 µL	1.489 mL	2.979 mL
10 mM	14.894 µL	74.469 µL	148.938 µL	744.69 µL	1.489 mL

Molarity calculator

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

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	102000¹
Excitation (nm)	642
Emission (nm)	657
Quantum yield	0.11¹

Product Family

Name	Excitation (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	515	531	62800¹	0.25¹
PRO-TAO-3 [equivalent to TO-PRO-3]	642	657	102000	0.11¹

Images

Figure 1. Chemical structure for TWO-PRO™ 3 [equivalent to TO-PRO®-3] *5 mM DMSO Solution* *CAS 157199-63-8*

Citations

View all 1 citations: Citation Explorer

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

References

View all 42 references: Citation Explorer

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

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)