Cy7: a reliable fluorescent dye for sensitive detection

Cyanine 7 (Cy7) is a cyanine dye which is fluorescent active in the near-infrared (NIR) region (emission ~ 770 nm). It is known for its high fluorescence, a feature of all cyanine dyes, in combination with a significantly lower photostability than other cyanine dyes. For a higher quantum yield the methine chains of almost all commercially available variants are modified with non-aromatic ring systems. Cy7 is used for biochemical or bioanalytical applications because of its near-infrared fluorescence emission in an area where biological specimens exhibit minimal autofluorescence. The low energy of the needed light for excitation of these dyes condition a minimal phototoxicity of the used light. All of these advantages are also available for far-red dyes as Cy5 and lead to an improved signal-to-noise ratio making Cy7 and Cy5 ideal dyes for a wide range of biological applications such as fluorescence in situ hybridization (FISH), RNA labeling or protein conjugation. If no multiplexing experiments with another far-red absorption dye is scheduled, usually Cy5 is superior due to higher photostability and a higher quantum yield.

Typical uses of Cy7 in life sciences

Cy7 can be used for labeling a variety of biomolecules for different applications. Due to its NIR properties it is an ideal candidate for multiplexing with other dyes in the visible light range.

• Antibody labeling: Antibodies could be labelled with Cy7 to detect or quantify proteins or antigens in complex living systems. The antibody protein complexes are subsequently analyzed by immunofluorescence or sorted by flow cytometry.
• Nucleic acid detection: Labeling of nucleic acids enables the detection of specific DNA or RNA sequences. This is used in fluorescence induced in situ hybridization (FISH) or for real-time PCR.
• Cell based assays: Cy7 is used to label cells or cellular components allowing to monitor cellular processes such as cell proliferation, apoptosis, or signal transfection. Due to its NIR absorbance, this dye is especially suited to the minimal autofluorescence in this spectral range of cells as well as the low phototoxicity for the used energy-poor light.
• In vivo imaging: The near-infrared properties of this dye make it an ideal candidate for in vivo imaging experiments since NIR photons can penetrate tissues more effectively than the one in the visible range. Therefore, Cy7 can be used for non-invasive imaging of biological processes in living organisms such as the tracking of drugs, monitoring of tumors, or studying disease progression.
• Flow cytometry: It is used for flow cytometry experiments since this dye can be excited with common lasers.

How Cy7 supports modern fluorescence applications

Long-wavelength signal and low background

Cy7 is a fluorescent dye with absorbance and emission in the far-red to near-infrared spectrum. Using a dye being fluorescence active in this region of the spectrum has the main advantage of reduced background to noise ratio due to the minimal autofluorescence of biological samples in the far-red to near-infrared region. Therefore, the use of near-infrared fluorophores enhances clarity and precision of imaging recordings. This enables the usage of Cy7 for complex biological samples where a high signal to noise ratio is mandatory. Since Cy7 dyes need to be handled with great care because of their low photostability, far-red dyes are often the better choice.

Ideal for multiplex labeling

Due to the near infra-red (NIR) properties, this dye can be combined with several other dyes with different absorption ranges without interfering with them. Especially, if high content readouts should be performed, a dye in the NIR (Cy7) or far-red (Cy5) spectra should be chosen, because they offer the best signal to noise ration due to minimal autofluorescence of biological samples at the needed wavelengths. Another advantage is deep tissue penetration of light in these wavelengths induced by minimal interactions with biological matter.

Combining Cy7 with Click Chemistry labeling

Cy7 is a dye that is stable under a variety of conditions for chemical labeling reactions. The one with the best incorporation rates is the alkyne azide cycloaddition. Here, the yield is the best for either the copper catalyzed- (CuAAC) or the strain promoted- (SPAAC) variants which are both known as click chemistry reactions. Therefore, if there is no explicit need for NIR dyes, far-red dyes usually provide the same advantages as for autofluorescence background in combination with easier and more efficient reaction.

The highly efficient click chemistry reaction is used for labeling of a wide variety of biomolecules including DNA, RNA, proteins, and antibodies.

Cy7 in flow cytometry and microscopy

Near infra-red Cy7 dyes are usable in the far-red channel of most flow cytometers as alternatives to the far-red dyes as Cy5 dyes. For flow cytometry lots of pre-conjugated variants with antibodies or streptavidin are available. Even tandem dyes such as PE-Cy7 or APC-Cy7 are available on the market. For the tandem dyes, the brightness of a donor dye is combined with the NIR emission of Cy7.

This dye is also used in multicolor panels because its NIR properties are well-separated from other fluorophores which leads to minimal spectral overlap.

There are additional usages in microscopy such as deep tissue imaging and live animal imaging. These techniques in turn take advantage of the special properties of Cy7, namely the deep penetration into tissue of NIR photons and the low autofluorescence of biological materials at the wavelengths used. The spectral properties again allow for a wide range of possible combinations with other fluorophores for multiplexing imaging experiments.

Practical tips for using Cy7

Cy7 dyes are relatively photo instable in comparison to other cyanine dyes. Therefore, they should be stored in a cold (-20 °C) and dark environment. Their stability can be enhanced if they are handled under low-light conditions such as using amber vials or dimmed environments. When multiplexing experiments are performed, the experiment with Cy7 dyes should be conducted before other fluorophores are analyzed.

For all these tasks Cy5 based far-red dyes can be used combining the same advantages in deep penetration and low autofluorescence of tissue combined with higher photostability and incorporation rates. Therefore, working with Cy5 dyes as Eterneon red 645 azide or DBCO-AF647 reduces the need for light protection during your sample preparation. For chemical labeling reactions such as click chemistry (CuAAC or SPAAC) Cy5 dyes usually provide better results than Cy7 dyes since they are chemically more stable and show better solubility.

Explore baseclick’s Cy7-compatible products and reagents

baseclick is providing a variety of different dyes in different wavelengths. All of them provide high photostability and brightness. All of them are available with functional moieties enabling click chemistry labeling reactions.