These five spectrally distinct monomeric cyanine nucleic acid stains are high sensitivity fluorescent probes for nucleic acid staining. The carbocyanine monomers have very strong binding affinity for dsDNA, with dissociation constants in the micromolar range. These dyes each comprise a single cyanine dye and a cationic side chain. The carbocyanine monomers are spectrally analogous to the corresponding dimeric cyanine dyes; however, with only two positive charges and one intercalating unit, these dyes exhibit somewhat reduced affinity for nucleic acids relative to the cyanine dimers.

Features of the monomeric cyanine nucleic acid stains:
• High affinity—strong binding affinity for dsDNA with dissociation constants in the micromolar range
• Low background—negligible fluorescence when not bound to nucleic acids
• Large fluorescence enhancement—20- to 1800-fold fluorescence enhancements upon binding to DNA
• Bright fluorescence—high extinction coefficients and fluorescence quantum yields resulting in very bright fluorescence signals

Like their dimeric counterparts, these monomeric cyanine dyes are typically impermeant to viable cells and can be used as dead cell indictors, although YO-PRO-1 (Cat. No. Y3603) can be permeant to apoptotic cells, providing a convenient indicator of apoptosis. The monomeric cyanine stains have wide applicability due to their low background and bright fluorescence. Uses include staining of nucleic acids on solid supports, prestaining of samples for gel or capillary electrophoresis, viability detection, and counterstaining in multiple-label experiments.

The fluorescence spectra of the five monomeric cyanine nucleic acid stains cover the entire visible wavelength range and each dye differs in extinction coefficient and quantum yield. These dyes may also be used with ultraviolet trans- or epi-illuminator excitation sources. TO-PRO-1, PO-PRO-1, and YO-PRO-1 dyes exhibit secondary ultraviolet excitation peaks near 275 nm. The monomeric cyanine nucleic acid stains exhibit large degrees of fluorescence enhancement upon binding to DNA (or RNA), varying from 400- to 1800-fold for the “1-series” and 20- to 200-fold for the “3-series.” Consequently, the fluorescence of unbound dye is negligible under most experimental detection conditions.

The monomeric cyanine nucleic acid stains are supplied in a unit size of 1 mL as 1 mM solutions in dimethylsulfoxide (DMSO).

PO-PRO™-1 Iodide (435/455)
The blue-fluorescent PO-PRO-1 stain has excitation/emission maxima ∼435/455 nm when bound to nucleic acid. It can be excited by ultraviolet (UV) excitation sources and the He-Cd 442 nm laser.

YO-PRO™-1 Iodide (491/509)
The green-fluorescent YO-PRO-1 stain has excitation/emission maxima ∼491/509 nm when bound to nucleic acid. It can be excited by ultraviolet (UV) excitation sources and the Ar 488 nm laser. YO-PRO-1 stain has been used to identify apoptotic cells. Apoptotic cells become permeant to YO-PRO-1, but remain impermeant to propidium iodide (Cat. No. P3566) and other dead cell stains. Live cells are not stained with YO-PRO-1, allowing them to be used in subsequent experiments. The YO-PRO-1 nucleic acid stain is also included as an apoptosis indicator in the Vybrant Apoptosis Assay Kit #4 (Cat. No. V13243) and Chromatin Condensation/Membrane Permeability/Dead Cell Apoptosis Kit (Cat. No. V23201).

TO-PRO™-1 Iodide (515/531)
The green-fluorescent TO-PRO-1 stain has excitation/emission maxima ∼515/531 nm when bound to nucleic acid. It can be excited by ultraviolet (UV) excitation sources and the Ar 514 nm laser.

YO-PRO™-3 Iodide (612/631)
The orange-fluorescent YO-PRO-3 stain has excitation/emission maxima ∼612/631 nm when bound to nucleic acid. It can be excited by the He-Ne 594 nm laser.

TO-PRO™-3 Iodide (642/661)
The far-red-fluorescent TO-PRO-3 stain has excitation/emission maxima ∼642/661 nm with fluorescence similar to Alexa Fluor 647 or Cy5 dyes. The long-wavelength fluorescence of TO-PRO-3 stain is well separated from that of commonly used fluorophores, such as the Alexa Fluor dyes, Oregon Green, fluorescein (FITC), rhodamine (TRITC), Texas Red, coumarin (AMCA), Marina Blue, and Pacific Blue dyes. Additionally, long-wavelength light–absorbing dyes such as TO-PRO-3 stain have the advantage that their fluorescence is usually not obscured by the autofluorescence of tissues.

TO-PRO-3 stain is useful as a nuclear counterstain and dead cell indicator in fluorescence microscopy and flow cytometry, and is among the highest-sensitivity probes for nucleic acid detection. TO-PRO-3 gives strong and selective staining of the nucleus in cultured cells and in paraffin sections. Simultaneous labeling with a green-fluorescent SYTO dye and cell-impermeant TO-PRO-3 stain is frequently used to assess cell viability. TO-PRO-3 has a much higher extinction coefficient than DNA-bound propidium iodide and has been used for unique applications such as detecting microbial cells and microorganisms in soil environments.

TO-PRO-3 is also available as room-temperature-stable, ready-to-use solutions:
• NucRed™ Dead 647 ReadyProbes™ Reagent (Cat. No. R37113)
• TO-PRO™ 3 Ready Flow™ Reagent (Cat. No. R37170)