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Cy3 dye

Manufactured by GE Healthcare
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Cy3 dye is a fluorescent labeling agent commonly used in various biomedical research applications. It emits a bright red-orange fluorescence when excited at the appropriate wavelength. The dye is often used in techniques such as fluorescence microscopy, Western blotting, and microarray analysis to label and visualize target biomolecules.

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Lab products found in correlation

Cy5 dye, by GE Healthcare (4 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (4 mentions) Feature extraction software, by Agilent Technologies (3 mentions) Rnaqueous micro kit, by Thermo Fisher Scientific (2 mentions) Sureprint g3 mouse gene expression 8x60k microarray, by Agilent Technologies (2 mentions) Aminoallyl messageamp 2 kit, by Thermo Fisher Scientific (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Akta chromatography system, by GE Healthcare (1 mentions) Ultracel 100k cellulose column, by Merck Group (1 mentions) Ni nta bead, by Qiagen (1 mentions) Facsaria, by BD (1 mentions) 5 n ethyl n isopropyl amiloride eipa, by Merck Group (1 mentions) Anti acetylated tubulin, by Merck Group (1 mentions) Transferrin alexa fluor 568, by Thermo Fisher Scientific (1 mentions) Ipa 3, by Merck Group (1 mentions) Cytochalasin b, by Merck Group (1 mentions) Lamp1, by Santa Cruz Biotechnology (1 mentions) Bafilomycin a1, by Merck Group (1 mentions) Cytochalasin d, by Merck Group (1 mentions)

23 protocols using cy3 dye

1

Cell Surface Glycan Profiling Using Lectin Microarray

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A lectin microarray platform was used to profile 45 different glycan structures on the surface of sorted cells (Hirabayashi et al., 2013 (link), 2015 (link); Nagahara et al., 2008 (link); Tateno et al., 2007 (link), 2009 (link), 2010 (link), 2011 (link); Uchiyama et al., 2008 (link)). The lectin array employs a representative panel of immobilized lectins with known glycan structure binding specificity. Cell-membrane proteins were labeled with Cy3 dye (GE Healthcare, cat# GEPA23001) and hybridized to the lectin microarray. The resulting lectin chips were scanned for fluorescence intensity on each lectin-coated spot using an evanescent-field fluorescence scanner (GlycoTechnica Ltd.). All samples were run in triplicate, and the average of the triplicate was used for analysis. Data were normalized using the global normalization method.
Colomb F., Giron L.B., Kuri-Cervantes L., Adeniji O.S., Ma T., Dweep H., Battivelli E., Verdin E., Palmer C.S., Tateno H., Kossenkov A.V., Roan N.R., Betts M.R, & Abdel-Mohsen M. (2020). Sialyl-LewisX Glycoantigen Is Enriched on Cells with Persistent HIV Transcription during Therapy. Cell reports, 32(5), 107991.
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2

Transcriptional Profiling of Mouse Embryos

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Labelled RNA samples were prepared as described0 (link). Total RNA was extracted with TRIzol (Life Technologies, Carlsbad, CA, USA) from 10 cloned 2-cell embryos cultured for 24 h. RNA was subjected to two rounds of linear amplification using TargetAmp Two-Round Aminoallyl-aRNA Amplification Kits (Epicentre, Madison, WI, USA) according to the manufacturer’s instructions. For oocytes, control 2-cell embryos and nuclear donor cells, 10 pooled MII oocytes, 10 pooled IVF-derived 2-cell embryos and 500 cumulus cells were all subjected to total RNA extraction and two rounds of RNA amplification. Amplified RNA was labelled with Cy3 dye (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and hybridized to a Whole Mouse Genome oligo DNA microarray (4 × 44 K; Agilent Technologies) for 16 h at 65 °C according to the manufacturer’s instructions.
Inoue K., Oikawa M., Kamimura S., Ogonuki N., Nakamura T., Nakano T., Abe K, & Ogura A. (2015). Trichostatin A specifically improves the aberrant expression of transcription factor genes in embryos produced by somatic cell nuclear transfer. Scientific Reports, 5, 10127.
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3

Overexpression and Purification of Biotinylated BNCs

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As described previously [20 (link),33 (link),34 (link)], BNCs were overexpressed in Saccharomyces cerevisiae AH22R− cells that carried the ZZ-BNC expression plasmid, pGLD-ZZ50. Next, as described previously [20 (link),35 (link)], BNCs were extracted by disrupting the cells with glass beads; then, BNCs were purified on an AKTA chromatography system (GE Healthcare). Next, BNCs were biotinylated with the EZ-Link Sulfo-N-hydroxysuccinimide-biotin kit (Pierce), according to the manufacturer’s protocol. For binding assays, biotinylated BNCs were labeled with Cy3-dye (GE Healthcare) with N-hydroxysuccinimide chemistry, as described previously [36 (link)]. Finally, the drug-containing, biotinylated BNC-LP complex was prepared by conjugating the biotinylated BNC with drug-containing liposomes at a weight ratio of 1:35, as described previously [17 (link)].
Yoshimura K., Aoki H., Teruyama C., Iijima M., Tsutsumi H., Kuroda S, & Hamano K. (2020). A Novel Hybrid Drug Delivery System for Treatment of Aortic Aneurysms. International Journal of Molecular Sciences, 21(15), 5538.
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4

Lectin Microarray Analysis of Salivary Glycoproteins

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A lectin microarray was obtained by synthesising 37 lectins with different binding preferences for the N and O chains (32 (link)). Salivary proteins were labelled with Cy3 dye (GE Healthcare, Boston, MA, USA). Cy3-labelled salivary proteins measuring 4 μg was mixed with 120 μg of incubation buffer and applied to lectin microarrays for 3 h at 37°C. Only sugar chains with a specific structure can bind to the corresponding lectin. Therefore, the fluorescence intensity of lectin represents the expression level of the corresponding glycoprotein.
Han Q., Wang X., Ding X., Hao J., Li Q., Wang J., Yu H., Tang Z., Yang F., Cai G., Zhang D, & Zhu H. (2022). Salivary Glycopatterns as Potential Non-Invasive Biomarkers for Diagnosing and Reflecting Severity and Prognosis of Diabetic Nephropathy. Frontiers in Endocrinology, 13, 790586.
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5

Immunofluorescence Assay with Cytoskeleton Markers

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Anti-α-tubulin (1:5000, cat. no. T5168), anti-acetylated tubulin (0.05μg/ml, cat. no. T7451), and chemicals, including cytochalasin D (cat. no. C8273), bafilomycin A1 (cat. no. 196000), nocodazole (cat. no. M1404) and streptolysin O (SLO; cat. no. S5265) were purchased from Sigma-Aldrich (Louise, MO). Antibodies to HA (1:5000, cat. no. AB9110), dynamin 2 (1:1000, cat. no. AB65556), and pericentrin (1:1000, cat. no. AB4448) were obtained from Abeam (Cambridge, MA). Anti-actin antibody (1:5000, cat. no. AM-4302) and EdU (5-ethynyl-2′-deoxyuridine) dye (cat. no. A10044) were obtained from Thermo Fisher Scientific (Waltham, MA). Anti-GM130 (1:1000, cat. no. 610822) was obtained from BD Transduction Laboratories (San Jose, CA). Antibodies to GAPDH (1:3000, cat. no. sc-322233) and Cy3 (1:200, cat. no. sc-166894) were obtained from Santa Cruz Biotechnology (Dallas, TX). Cy3 dye was obtained from GE Healthcare Life Sciences (Pittsburgh, PA, cat. no. PA23031). Lipofectamine® RNAiMAX ((Thermo Fisher Scientific, cat. no. 13778500)) and Lipofectamine® LTX Reagent with PLUS™ Reagent (cat. no. 15338030) were used for siRNAs transfection and plasmids transfection, respectively.
Lee J.S., Ismail A.M., Lee J.Y., Zhou X., Materne E.C., Chodosh J, & Rajaiya J. (2019). Impact of dynamin 2 on adenovirus nuclear entry. Virology, 529, 43-56.
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6

Purification and Labeling of Cas9 Protein

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After transformation of the pET28-NLS-Cas9 vector into E. coli strain BL21 (DE3), Cas9 protein expression was induced for 12 h at 18°C with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The bacterial cells were lysed by sonication; after centrifugation at 20,000g for 30 min, the soluble lysate was mixed with Ni-NTA beads (Qiagen), and Cy3 dye (GE Healthcare) was added at a 1:10 ratio (protein:dye molecules). The mixture was incubated overnight (>12 h) at 4°C in the dark. Cy3-labeled Cas9 was eluted with elution buffer (50 mM Tris-HCl [pH 7.6], 150–500 mM NaCl, 10%–25% glycerol, 0.2 M imidazole) and dialyzed against dialyzing buffer (20 mM HEPES pH 7.5, 150 mM KCl, 1 mM DTT, 10% glycerol). The purified Cy3-labeled Cas9 protein was concentrated using an Ultracel 100K cellulose column (Millipore). The purity of the Cy3-labeled Cas9 protein was determined by SDS-PAGE. The Cy3 labeling efficiency was measured by comparing the absorption spectra of the Cas9 protein (280 nm) and the conjugated Cy3 dye molecule.
Kim K., Park S.W., Kim J.H., Lee S.H., Kim D., Koo T., Kim K.E., Kim J.H, & Kim J.S. (2017). Genome surgery using Cas9 ribonucleoproteins for the treatment of age-related macular degeneration. Genome Research, 27(3), 419-426.
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7

Fluorescent Labeling of HEp-2 Proteins

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This step was exclusively used for the FBIP approach. Fifty micrograms per strip of HEp-2 extract protein were labeled with 400 pmol Cy3 dye (GE Healthcare), according the manufacturer’s instructions. Cy5 dye labeling was used when a post-staining step with Deep Purple (DP, GE Healthcare) was performed.
Dutoit-Lefèvre V., Dubucquoi S., Launay D., Sobanski V., Dussart P., Chafey P., Broussard C., Duban-Deweer S., Vermersch P., Prin L, & Lefranc D. (2015). An Optimized Fluorescence-Based Bidimensional Immunoproteomic Approach for Accurate Screening of Autoantibodies. PLoS ONE, 10(7), e0132142.
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8

Labeling and Profiling Membrane Proteins

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Membrane proteins extracted from CD8+ and NK cells were labeled with Cy3 dye (GE Healthcare, catalogue# GEPA23001) and hybridized to the lectin microarrays. The resulting chips were scanned for fluorescence intensity on each lectin-coated spot using an evanescent-field fluorescence scanner GlycoStation Reader (GlycoTechnica Ltd.), and data were normalized using the global normalization method.
Giron L.B., Colomb F., Papasavvas E., Azzoni L., Yin X., Fair M., Anzurez A., Damra M., Mounzer K., Kostman J.R., Tebas P., O'Doherty U., Tateno H., Liu Q., Betts M.R., Montaner L.J, & Abdel-Mohsen M. (2020). Interferon-α alters host glycosylation machinery during treated HIV infection. EBioMedicine, 59, 102945.
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9

Cell Surface Glycan Profiling Using Lectin Microarray

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A lectin microarray platform was used to profile 45 different glycan structures on the surface of sorted cells (Hirabayashi et al., 2013 (link), 2015 (link); Nagahara et al., 2008 (link); Tateno et al., 2007 (link), 2009 (link), 2010 (link), 2011 (link); Uchiyama et al., 2008 (link)). The lectin array employs a representative panel of immobilized lectins with known glycan structure binding specificity. Cell-membrane proteins were labeled with Cy3 dye (GE Healthcare, cat# GEPA23001) and hybridized to the lectin microarray. The resulting lectin chips were scanned for fluorescence intensity on each lectin-coated spot using an evanescent-field fluorescence scanner (GlycoTechnica Ltd.). All samples were run in triplicate, and the average of the triplicate was used for analysis. Data were normalized using the global normalization method.
Colomb F., Giron L.B., Kuri-Cervantes L., Adeniji O.S., Ma T., Dweep H., Battivelli E., Verdin E., Palmer C.S., Tateno H., Kossenkov A.V., Roan N.R., Betts M.R, & Abdel-Mohsen M. (2020). Sialyl-LewisX Glycoantigen Is Enriched on Cells with Persistent HIV Transcription during Therapy. Cell reports, 32(5), 107991.
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10

Antigen-experienced T cell transcriptional profiling

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Antigen-experienced T cells (S2A Fig, [42 ,43 ]) were isolated from wild-type mice 6 d.p.i. by double-sorting to high purity on a FACSAria (BD). RNA was isolated using the RNAqueous Micro kit (Ambion) and amplified (Amino Allyl MessageAmp II kit, Life Technologies) to generate amino allyl incorporated amplified RNA (aaRNA). aaRNA was coupled to Cy3 dye (GE Healthcare Life Sciences) and hybridized overnight to a SurePrint G3 Mouse Gene Expression 8x60K microarray (Agilent), which was washed and scanned per manufacturer’s instructions. Raw intensities were extracted using Feature Extraction software (Agilent) and quantile normalized using Limma [87 ]. Differentially expressed genes were identified using Significance Analysis for Microarrays (SAM) [88 (link)]. Complete microarray data can be accessed in the Gene Expression Omnibus database (GEO; http://www.ncbi.nlm.nih.gov/geo/) under accession GSE81196.
Fontana M.F., de Melo G.L., Anidi C., Hamburger R., Kim C.Y., Lee S.Y., Pham J, & Kim C.C. (2016). Macrophage Colony Stimulating Factor Derived from CD4+ T Cells Contributes to Control of a Blood-Borne Infection. PLoS Pathogens, 12(12), e1006046.
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