Chemical & Physical Differences

Sulfo-Cyanine5 and Cy5 NHS Ester are both cyanine-based fluorescent dyes widely used in biological imaging, but they behave very differently due to their chemical structures.

Sulfo-Cyanine5 is a sulfated version of Cyanine5, meaning it contains two negatively charged sulfonate groups in its structure [1]. These groups significantly boost the dye’s water solubility and hydrophilicity [2][3]. This makes Sulfo-Cyanine5 ideal for working in aqueous environments, without needing organic solvents [2][3].

In contrast, Cy5 NHS Ester is strongly hydrophobic [4]. It lacks the sulfonate modifications, making it more lipophilic and prone to associating with lipid membranes or cellular organelles [4][5].

Spectral Properties

Despite structural differences, both dyes share nearly identical spectral features, as they come from the same core fluorophore:

• Excitation maximum: ~646–649 nm [2][3][5]
• Emission maximum: ~662–670 nm [2][3][5]
• High extinction coefficient: ~250,000–271,000 M⁻¹cm⁻¹ [2][3][5]

They also show comparable fluorescence brightness and quantum yields [3][5].

Cell Membrane Permeability: The Deal-Breaker

One of the most important distinctions for live cell imaging is how these dyes interact with cell membranes:

• Sulfo-Cyanine5 is cell-impermeable. Its charged sulfonate groups remain ionized at physiological pH, preventing the dye from crossing intact plasma membranes [6][7]. This makes it perfect for cell surface labeling, but not usable for labeling inside live cells [6][7].
• Cy5 NHS Ester, being hydrophobic, shows different permeability characteristics. While it can sometimes penetrate cells, it often sticks nonspecifically to lipid-rich areas like the endoplasmic reticulum or Golgi apparatus, leading to high background staining [4][8]. Its permeability varies depending on formulation and use conditions [5].

Suitability for Live Cell Tracing

Unfortunately, neither dye is ideal for live cell tracing in their standard forms:

Sulfo-Cyanine5:

• Can’t cross membranes [6][7]
• Limited to external surface labeling [6][7]

Cy5 NHS Ester:

• Hydrophobic nature causes nonspecific binding [4]
• May accumulate in intracellular membranes and cause unwanted background signals [8]

✅ Better Alternatives for Live Cell Tracing

For more reliable live cell imaging, you should consider:

Toxicity Note

Regardless of the dye, concentration matters. High doses can be toxic and compromise cell viability [11][13]. Interestingly, Sulfo-Cyanine5 may be safer, as it stays outside the cell and is less likely to interfere with internal processes [6].

Sources are taken from perplexity pro

[1] sulfo-Cyanine5 hydrazide | CAS#:2055138-61-7 - Lumiprobe https://www.lumiprobe.com/p/sulfo-cy5-hydrazide

[2] Sulfo-Cyanine5 NHS ester - CAS 2230212-27-6 https://www.santiago-lab.com/product/sulfo-cyanine5-nhs-ester-cas-2230212-27-6/

[3] Sulfo-Cyanine5 NHS ester https://probes.bocsci.com/product/sulfo-cyanine5-nhs-ester-426839.html?nid=7548

[4] Is Cy5 hydrophobic? https://www.aatbio.com/resources/faq-frequently-asked-questions/Is-Cy5-hydrophobic

[5] Cyanine 5, SE - Fluorescent Dyes - Tocris Bioscience https://www.tocris.com/products/cyanine-5-se_5436

[6] Sulfonated red and far-red rhodamines to visualize SNAP https://pmc.ncbi.nlm.nih.gov/articles/PMC9346974/

[7] Sulfonated rhodamines as impermeable labelling substrates for cell ... https://www.biorxiv.org/content/10.1101/2021.03.16.435698.full

[8] bc400575w 1..9 https://pubs.acs.org/doi/pdf/10.1021/bc400575w

[9] Live Cell Imaging Reagents - Sigma-Aldrich https://www.sigmaaldrich.com/US/en/products/cell-culture-and-analysis/cell-analysis/live-cell-imaging-reagents

[10] Live cell staining dyes - Abcam https://www.abcam.com/en-us/technical-resources/product-overview/live-cell-staining-dyes

[11] Data acquisition and Cell sorting Section V https://wi.mit.edu/sites/default/files/2021-05/20200402_Vital_Dyes_l_(1).pdf

[12] MP 03224 https://tools.thermofisher.com/content/sfs/manuals/mp03224.pdf

[13] A cytotoxicity assay utilizing a fluorescent dye that ... - PubMed https://pubmed.ncbi.nlm.nih.gov/8034974/

[14] Cytiva Cy5.5 NHS Ester Mono-Reactive CyDye - Protein Analysis Reagents, Crosslinking, Labeling and Protein Modification https://www.fishersci.co.uk/shop/products/cy5-5-nhs-ester-mono-reactive-cydye/11585085

[15] CY5 (Sulfo-Cyanine5) | Reactive Dye - MedchemExpress.com https://www.medchemexpress.com/CY5.html

[16] Cy5 NHS ester(Et) https://www.apexbt.com/cy5-nhs-ester-et.html

[17] Sulfo-Cy5 amine | Fluorochrome Dye - MedchemExpress.com https://www.medchemexpress.com/sulfo-cy5-amine.html

[18] [PDF] Cy5-NHS Ester Technical Information | GoldBio https://goldbio.com/documents/2536/CY5-NHS%20Ester%20Technical%20Information%20Final.pdf

[19] Live Cell Fluorescent Organelle Dyes and Stains - Sigma-Aldrich https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/imaging-analysis-and-live-cell-imaging/organelle-dyes-and-stains

[20] Effect of Vital Dyes on Retinal Pigmented Epithelial Cell Viability and Apoptosis: Implications for Chromovitrectomy https://karger.com/oph/article-abstract/230/Suppl.%202/41/264046/Effect-of-Vital-Dyes-on-Retinal-Pigmented?redirectedFrom=fulltext

[21] Mechanism of Cyanine5 to Cyanine3 Photoconversion and Its ... https://pubs.acs.org/doi/10.1021/jacs.1c04178

[22] Comparative Analysis of the Different Dyes' Potential to Assess Human Normal and Cancer Cell Viability In Vitro under Different D/H Ratios in a Culture Medium. https://go.gale.com/ps/i.do?asid=5e98b242&id=GALE%7CA622651289&it=r&p=AONE&sid=googleScholar&u=googlescholar&v=2.1

[23] Sulfo-Cyanine 5 NHS ester https://www.aatbio.com/products/sulfo-cyanine-5-nhs-ester

[24] CY5-SE (Cy5 NHS Ester) | Reactive Dye - MedchemExpress.com https://www.medchemexpress.com/CY5-SE.html

[25] [PDF] A one-step protocol to generate impermeable fluorescent HaloTag ... https://www.biorxiv.org/content/10.1101/2024.09.20.614087v1.full.pdf

[26] Products https://nordicbiosite.com/products?sortBy=size&sortDirection=asc&search_text=K025-H&excludeName=application.keyword&excludeValue=MS&filterName=displaySupplierName.keyword&filterValue=MedChemExpress&offset=3&limit=50

[27] Live-Cell Imaging | Thermo Fisher Scientific - UK https://www.thermofisher.com/uk/en/home/life-science/cell-analysis/cellular-imaging/fluorescence-microscopy-and-immunofluorescence-if/microscopy-reagents-and-media/live-cell-imaging-reagents.html

[28] A cytotoxicity assay utilizing a fluorescent dye that determines accurate surviving fractions of cells. https://www.sigmaaldrich.com/MH/en/tech-docs/paper/98866

[29] Best practices: 5 steps to live-cell imaging - YouTube https://www.youtube.com/watch?v=bbhd_SqqLBk

[30] A Guide to Fluorescent Dyes in Life Science Research - Labinsights https://labinsights.nl/en/article/a-guide-to-fluorescent-dyes-in-life-science-research

[31] LIVE/DEAD Cell Viability Assays | Thermo Fisher Scientific - US https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/cell-viability/live-dead-cell-viability-assays.html

[32] Laurdan in live cell imaging: Effect of acquisition settings, cell culture ... https://www.sciencedirect.com/science/article/pii/S1011134422000185

[33] https://www.ibiantech.com/catalogos/lumiprobe/Sulfo-Cyanine5%20NHS%20ester.pdf

[34] Sulfo-Cy5 amine - MedChem Express - Cambridge Bioscience https://www.bioscience.co.uk/product~1662747

[35] In vitro cellular toxicity of ILM dyes on human RPE and Müller glia cell lines https://iovs.arvojournals.org/article.aspx?articleid=2769334

[36] Live-Cell Imaging | Thermo Fisher Scientific - US https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cellular-imaging/fluorescence-microscopy-and-immunofluorescence-if/microscopy-reagents-and-media/live-cell-imaging-reagents.html

[37] A membrane-permeable dye for living cells with large two-photon excited fluorescence action cross-sections for bioimaging https://pubs.rsc.org/en/content/articlelanding/2015/tb/c5tb00940e/unauth

[38] Cell Viability Assay Kit, Green/Red Fluorescence https://www.stemcell.com/products/cell-viability-assay-kit-green-red-fluorescence.html

Sulfo-Cyanine5 (Cy5) is the best option.

Because it is more water-soluble and less toxic, it can be used directly to track live cells without additional chemical reactions.

The difference in their chemical composition and the presence of amine and ester groups according to their biological and physiological functions in living organisms.

Both products are available from MedChemExpress (MCE), with detailed biological activities documented on the product detail pages: Sulfo-Cyanine5 (HY-D0821, MCE) and Cy5 NHS Ester (HY-D0819, MCE).

Information can be obtained by searching the catalog number on the official website.

The primary difference between Sulfo-Cyanine5 (Cy5) and Cy5 NHS Ester lies in their reactive chemical groups. HY-D0821 is the non-activated Sulfo-Cyanine5, which requires additional activation steps for conjugation with biomolecules. In contrast, HY-D0819 is the succinimidyl ester (SE)-activated Cy5 NHS Ester, which can directly form covalent bonds with amine-containing molecules (e.g., proteins, peptides, etc.).

For live-cell tracking applications, HY-D0819 (Cy5 NHS Ester) is more suitable because it is pre-activated, enabling efficient labeling of amine groups on the cell surface or inside cells without requiring additional activation steps.

The answer to this question comes from MedChemExpress (MCE) Technical Support.

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