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Sulfo cyanine5 azide

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Sulfo-Cyanine5 azide is a fluorescent labeling reagent used in biological research. It is a cyanine dye with an azide functional group, which allows for the labeling of biomolecules through click chemistry reactions.

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Lab products found in correlation

5 ethynyl 2 deoxyuridine (edu), by Lumiprobe (2 mentions) Eclipse te2000 u inverted microscope, by Nikon (2 mentions) Click it rna imaging kit, by Thermo Fisher Scientific (1 mentions) Cuso4, by Merck Group (1 mentions) Sodium ascorbate, by Merck Group (1 mentions) Cyanine3 (cy3), by Lumiprobe (1 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions) 5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (1 mentions) Incarta software, by GE Healthcare (1 mentions) Gallios flow cytometer, by Beckman Coulter (1 mentions) Alexa fluor 594 goat anti mouse, by Thermo Fisher Scientific (1 mentions) Goat anti rabbit cy5, by Thermo Fisher Scientific (1 mentions) Alexa fluor 594 goat anti rabbit, by Thermo Fisher Scientific (1 mentions) Rabbit anti gapdh, by Cell Signaling Technology (1 mentions) Mouse anti tuj1, by Fortrea (1 mentions) Rabbit anti map2, by Cell Signaling Technology (1 mentions) Rabbit anti psd95, by Cell Signaling Technology (1 mentions) T6793, by Merck Group (1 mentions) Anhydrous sodium acetate, by Merck Group (1 mentions) Acetic anhydride, by Thermo Fisher Scientific (1 mentions)

12 protocols using sulfo cyanine5 azide

1

Dual DNA and RNA Labeling Methodology

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For nascent DNA labelling, cells were incubated with 40 μM EdU for 15 min before collection, fixed in 3.7% formaldehyde and permeabilized with 0.5% Triton X-100. EdU incorporation was revealed by a click-iT reaction: 4 mM CuSO4 (Merck Millipore), 2.5 μM Sulfo-cyanine 5 Azide (Lumiprobe), 100 mM Sodium Ascorbate (Merck Millipore). DNA was stained with DAPI, and coverslips were mounted.
For nascent RNA labelling, cells were incubated with 1 mM EU for 1 h and processed as above. EU incorporation was revealed with Click-iT® RNA Imaging kits (Thermo Fisher Scientific) using when indicated either Alexa Fluor 488 (Thermo Fisher Scientific) or a Cy3 (Lumiprobe) dyes Azides according to manufacturer's instructions. EU incorporation in the nucleolar compartment (identified by NPM1 staining) was assessed by multiplying the EU-positive nucleolar area (number of pixels) by the EU signal intensity (mean) in the NPM1-positive nucleolar compartment normalized to the EU signal intensity (mean) in the surrounding nucleoplasm, giving the so-called Integrated Density (IntDen).
Hernández‐Carralero E., Cabrera E., Rodríguez-Torres G., Hernández-Reyes Y., Singh A.N., Santa-María C., Fernández-Justel J.M., Janssens R.C., Marteijn J.A., Evert B.O., Mailand N., Gómez M., Ramadan K., Smits V.A, & Freire R. (2023). ATXN3 controls DNA replication and transcription by regulating chromatin structure. Nucleic Acids Research, 51(11), 5396-5413.
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2

Quantifying Cell Cycle Dynamics by EdU Imaging

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To analyze the percentage of cells in S phase an EdU imaging assay was performed. Briefly, semi-confluent cells were grown in black 96-well plates in the presence of 10 µM EdU (Thermo Fisher Scientific, C10635) for 60 min. After fixation in 4% w/v formaldehyde, permeabilization with 0.2% Triton X-100, EdU labeling with 4 mM Sulfo-Cyanine5 azide (Lumiprobe, A3330) using click chemistry and counterstaining with 5 µg/mL of Hoechst33342, fluorescence intensities for Cyanine5 and Hoechst in single nuclei was measured using an IN Cell 6500 and IN Carta software (GE Life Sciences, GE Healthcare, Chicago, IL, USA) and the ratio of EdU-positive to total nuclei per well was determined with R software.
Suraweera A., Duijf P.H., Jekimovs C., Schrobback K., Liu C., Adams M.N., O’Byrne K.J, & Richard D.J. (2021). COMMD1, from the Repair of DNA Double Strand Breaks, to a Novel Anti-Cancer Therapeutic Target. Cancers, 13(4), 830.
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3

Proliferation Assay with EdU

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Tumor cells or spleen cells were placed in culture for 24–72 hours. During the last 24 hours of the assay, EdU (Click-it assay, Thermo Fisher, Waltham, MA) was added to the cultures. After 24 hours, the cells were surface stained and/or permeabilized and stained with sulfo-cyanine5-azide (Lumiprobe, Hunt Valley, MD). Cell data was collected on a Gallios Flow Cytometer (Beckman Coulter, Brea, CA) and analyzed using FlowJo Software (FlowJo, Ashland, OR).
Park H., Gladstone M., Shanley C., Goodrich R, & Guth A. (2020). A Novel Cancer Immunotherapy Utilizing Autologous Tumor Tissue. Vox sanguinis, 115(6), 525-535.
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4

Immunostaining and EdU Labeling of Neural Cells

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Cells were fixed and immunostained as described(Huang et al. 2015 (link)). The following primary antibodies were used: rabbit anti-Neurog2 (1:1000; Santa Cruz; RRID: AB_2149513), rabbit anti-Map2 (1:1000; Cell Signaling Technology; RRID: AB_1069372), mouse anti-TuJ1 (1:1000; Covance; RRID: AB_2313773), rabbit anti-Syn (1:1000; Cell Signaling Technology; RRID: AB_2200102), rabbit anti-PSD95 (1:1000; Cell Signaling Technology; RRID: AB_2292883), rabbit anti-GAPDH (1:5000; Cell Signaling Technology; RRID: AB_16422005). Secondary antibodies used were goat anti-mouse Alexa Fluor 594 (1:1000; Invitrogen), goat anti-rabbit Alexa Fluor 594 (1:1000; Invitrogen), goat anti-mouse Cy5 (1:1000; Molecular Probes), goat anti-rabbit Cy5 (1:1000; Molecular Probes). For EdU staining, cells were pulsed with 1 mM EdU for 2.5h and visualized using the cocktail containing 100 mM Tris buffered saline, 4 mM CuSO4, 2 μM Sulfo-Cyanine 5 azide (Lumiprobe), and 100 mM sodium ascorbate. Nuclear counterstaining was performed with DAPI.
Gupta S., M-Redmond T., Meng F., Tidball A., Akil H., Watson S., Parent J.M, & Uhler M. (2017). Fibroblast Growth Factor 2 Regulates Activity and Gene Expression of Human Postmitotic Excitatory Neurons. Journal of neurochemistry, 145(3), 188-203.
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5

Labeling Proliferating Cells in Worms

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Proliferating cells were labeled by incubating the worms for 15 min with 5 µM EdU (5-ethynyl-2′-deoxyuridine, analogous to thymidine) just before anesthetization and fixation in PFA. After washes with PBS and one fast wash with 0.1 M Tris buffer (pH = 8.5), the samples were put in the click-reaction mixture for 45 min at room temperature. The mixture consisted of 100 mM Tris (pH = 8.5), 4 mM CuSO4, 2 µM sulfo-cyanine-5-azide (Lumiprobe), and 50 mM ascorbic acid in deionized water (I. Borisenko, personal communication). After this, the samples were rinsed with PBT and subjected to immunocytochemical detection of acetylated tubulin and nuclei labeling with DAPI. Acetylated tubulin was detected using primary antibodies (Sigma T6793, dilution 1:250) and fluorescent secondary antibody anti-mouse AF568 (Invitrogen, dilution 1:500) as previously described [3 (link)]. Incubation in antibodies took 1–2 days at +4 °C. After this, the samples were left in DAPI (1 µg/mL) for 1–2 h at room temperature or +4 °C overnight, then rinsed with PTW and stored in 90% glycerol.
F-actin labeling was performed independently of the other types of detection using phalloidin-AF488 (Invitrogen A12379, concentration 5 units/mL) as previously described [3 (link)]. The samples were incubated for 2 h at room temperature, then rinsed with phosphate buffer and stored in 90% glycerol.
Shalaeva A.Y., Kostyuchenko R.P, & Kozin V.V. (2021). Structural and Functional Characterization of the FGF Signaling Pathway in Regeneration of the Polychaete Worm Alitta virens (Annelida, Errantia). Genes, 12(6), 788.
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6

Tracking Neurogenesis in Developing Mouse Brain

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Pregnant mouse dams were injected at E13.5 with 1 mg EdU/10 g body weight, delivered as 10 mg/mL solution of 5-Ethynyl-2′-deoxyuridine (EdU, from Carbosynth) in 0.9% sodium chloride saline. Brain tissue was collected from P0 pups by anesthetizing pups, decapitation, 30 min immersion of the head in 4% PFA, and further processing/cryosectioning as outlined earlier in the protocol. To label EdU, slide-mounted sections were rinsed in PBS thrice (rinsed), permeabilized in PBS + 0.5% Triton X-100, rinsed, incubated for 5 min in CLICK reaction cocktail [4 mM CuSO4 pentahydrate, 5 μM Sulfo-Cyanine 5 Azide (Lumiprobe #A3330), 100 mM sodium ascorbate in Tris-buffered saline], rinsed, dried for 30 min, and mounted/coverslipped using Prolong Gold with DAPI.
Zavalin K., Hassan A., Fu C., Delpire E, & Lagrange A.H. (2022). Loss of KCC2 in GABAergic Neurons Causes Seizures and an Imbalance of Cortical Interneurons. Frontiers in Molecular Neuroscience, 15, 826427.
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7

Fullerene Functionalization and Labeling

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All of the chemicals used were of reagent-grade
quality or better, and the solvents were dried according to literature
procedures. The following reagents were used as received: C60 (99.5+%, SES Research, USA), propargyl alcohol (Acros Organics), p-toluenesulfonic acid monohydrate (Sigma-Aldrich), 1,8-diaza-bicyclo[5.4.0]undec-7-ene
(DBU, Sigma-Aldrich), malonic acid (Sigma-Aldrich), CBr4 (Sigma-Aldrich), 2-amino-1,3-propanediol (AK Scientific), acetic
anhydride (Fisher), N-acetylglycine (Acros Organics),
2,4-dihydroxybenzaldehyde (Acros Organics), anhydrous sodium acetate
(Sigma-Aldrich), sodium nitrite (Avantor), sodium azide (Sigma-Aldrich),
copper sulfate pentahydrate (Avantor), tris(2-carboxyethyl)phosphine
(Sigma-Aldrich), and sulfo-cyanine5 azide (Lumiprobe).
Serda M., Malarz K., Korzuch J., Szubka M., Zubko M, & Musioł R. (2022). In Situ Cellular Localization of Nonfluorescent [60]Fullerene Nanomaterial in MCF-7 Breast Cancer Cells. ACS Biomaterials Science & Engineering, 8(8), 3450-3462.
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8

Quantifying Cell Proliferation via EdU Assay

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Cell proliferation analysis was performed with the EdU click reaction containing 2Mm CuSO4.5H2O (Sigma), 8 µM Sulfo-Cyanine5 azide (Lumiprobe) and 20mg/ml Ascorbic acid (Sigma). For EdU incorporation, Col-0 plants were grown under standard conditions described above, and, at 7 days after plating, were transferred to ½ MS media supplemented with CNQX (50 mM) or mock. After 60 min pre-incubation, plants were placed on CNQX or mock cutting board, cut at 270 µm (high cuts) and immediately transferred to ½ MS medium containing CNQX (50 µM)/5 µM EdU or mock/5 µM EdU for 30min. Plants were fixed and permeabilized according to published protocols (Kazda et al., 2016 ). The next day, samples were washed with PBS twice and incubated in the EdU click reaction cocktail for 30 min in the dark. After two washes with PBS, plants were placed on 0.1mM DAPI in PBS and incubated for 30 min at 22 °C in the dark. Two final washes with PBS were performed. Samples were observed under an inverted fluorescence microscope. Images were analyzed with Image J software Fiji 1.52p version (http://imagej.nih.gov/ij/). Using images from a median optical section, labeled cells were quantified for EdU and DAPI in the meristematic zone. The boundary between the meristematic and the elongation zones is defined by a twofold or more increase in the distance between nuclei in the cortical cell layer.
Hernćndez-Coronado M., Dias Araujo P.C., Ip P.L., Nunes C.O., Rahni R., Wudick M.M., Lizzio M.A., Feijó J.A, & Birnbaum K.D. (2022). Plant glutamate receptors mediate a bet-hedging strategy between regeneration and defense. Developmental cell, 57(4), 451-465.e6.
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9

Quantifying Proliferation in Intestinal Epithelial Cells

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The 5-ethynyl-2-deoxyuridine (EdU)-based staining was performed on 2D-cultured primary IECs. Cells were sub-cultured from 48-well plates to 96-well plates at a ratio of 1:3, expanded in EM for 4 days followed by additional 2 days in EM with or without 10 ng/ml BMP9 and 100 ng/ml ALK1-Fc chimera protein or in DM. Cells were incubated with 10mM EdU for 3 hours at 37˚C. DNA-incorporated EdU was detected by Sulfo-Cyanine5azide (Lumiprobe, MD, USA) through a copper-catalyzed covalent reaction between an azide and alkyne22. Images were captured using a Nikon Eclipse TE2000-U inverted microscope (Nikon, Tokyo, Japan) and processed using NIS-Elements AR version 3.2 software. The EdU fluorescence area was measured by ImageJ software and normalized by the total cell area occupied by the Hoechst 33342 fluorescence as we described previously18.
Toyonaga T., Keith B.P., Barrow J., Schaner M.R., Wolber E.A., Beasley C., Huling J., Wang Y., Allbritton N.L., Chaumont N., Planche T., Koruda M.J., Rahbar R., Furey T.S., Sethupathy P., & Sheikh S.Z. (2020). Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier integrity and is associated with a poor clinical outcome in Crohn’s disease.
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10

Quantifying Proliferation in Intestinal Epithelial Cells

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The 5-ethynyl-2-deoxyuridine (EdU)-based staining was performed on 2D-cultured primary IECs. Cells were sub-cultured from 48-well plates to 96-well plates at a ratio of 1:3, expanded in EM for 4 days followed by additional 2 days in EM with or without 10 ng/ml BMP9 and 100 ng/ml ALK1-Fc chimera protein or in DM. Cells were incubated with 10mM EdU for 3 hours at 37˚C. DNA-incorporated EdU was detected by Sulfo-Cyanine5azide (Lumiprobe, MD, USA) through a copper-catalyzed covalent reaction between an azide and alkyne22. Images were captured using a Nikon Eclipse TE2000-U inverted microscope (Nikon, Tokyo, Japan) and processed using NIS-Elements AR version 3.2 software. The EdU fluorescence area was measured by ImageJ software and normalized by the total cell area occupied by the Hoechst 33342 fluorescence as we described previously18.
Toyonaga T., Keith B.P., Barrow J., Schaner M.R., Wolber E.A., Beasley C., Huling J., Wang Y., Allbritton N.L., Furey T.S., Sethupathy P., & Sheikh S. (2020). P149 DECREASED COLONIC ACTIVIN RECEPTOR-LIKE KINASE 1 DISRUPTS EPITHELIAL BARRIER INTEGRITY AND IS ASSOCIATED WITH A POOR CLINICAL OUTCOME IN CROHN’S DISEASE. Gastroenterology, 158(3), S46-S46.
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