Cy3 fluorescent dye

Manufactured by GE Healthcare

Sourced in United Kingdom

Cy3 is a fluorescent dye used in various laboratory applications. It is an orange-red fluorescent dye that is commonly used for labeling and detection purposes in techniques such as Western blotting, immunohistochemistry, and fluorescence microscopy.

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Lab products found in correlation

Sephadex g 25 column, by GE Healthcare (2 mentions) Genepix 4000b confocal scanner, by Molecular Devices (1 mentions) Cy5 fluorescent dye, by GE Healthcare (1 mentions) Sambucus nigra agglutinin, by Vector Laboratories (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions)

7 protocols using cy3 fluorescent dye

Saliva Collection Protocol for Biomarker Discovery

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Unstimulated saliva was collected between 9 a.m. and 10 a.m., at least 2 h after the last intake of food, from patients with T2DM, HB, HC, HCC and GC and from the healthy groups according to the protocol20 (link)29 (link)30 (link). The mouth was rinsed with physiological saline immediately before collection. Whole saliva (approximately 1 mL) was collected and placed on ice. Protease Cocktail Inhibitor (1 μL/mL of whole saliva) was added to the saliva immediately after collection to minimize protein degradation.
Whole saliva was then centrifuged at 12 000 rpm at 4°C for 30 min to remove the insoluble materials. The supernatant was collected and filtered with a 0.20-μm pore size to remove bacteria and microbials and then immediately used or stored at −80°C. To normalize the differences between subjects and to account for individual variation, 100 μL from each saliva sample was pooled in each group, the other maintained for further validation. The pooled saliva protein concentration of the T2DM, HB, HC, HCC and GC groups as well as the healthy groups determined by the Bradford assay (Supplementary Figure S1) were listed in Supplementary Table S1, S2 and S3. The pooled salivary proteins were then labeled with Cy3 fluorescent dye (GE Healthcare, Buckinghamshire, UK) and purified using Sephadex G-25 columns according to the manufacturer's instructions.

Zhong Y., Qin Y., Yu H., Yu J., Wu H., Chen L., Zhang P., Wang X., Jia Z., Guo Y., Zhang H., Shan J., Wang Y., Xie H., Li X, & Li Z. (2015). Avian Influenza Virus Infection Risk in Humans with Chronic Diseases. Scientific Reports, 5, 8971.

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Lectin Microarray Analysis of Salivary Glycoproteins

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The salivary proteins were labeled with Cy3 fluorescent dye (GE Healthcare, Buckinghamshire, UK) and purified using Sephadex G-25 columns according to the manufacturer's instructions. Subsequently, the Cy3-labeled salivary proteins were quantified and stored at − 20 °C in the dark until use. The lectin microarrays were produced as previously described (Qin et al., 2013 (link), Zhong et al., 2015 (link)). Briefly, 37 lectins with different binding preferences covering N- and O-linked glycans were spotted on homemade epoxysilane-coated slides. Each lectin was spotted in triplicate per block, with quadruplicate blocks on one slide. After immobilization, the slides were blocked with blocking buffer containing 2% BSA in 1 × PBS (0.01 mol/L phosphate buffer containing 0.15 mol/L NaCl, pH 7.4) for 1 h and rinsed twice with 1 × PBS. Then the blocked slide was incubated with Cy3-labeled salivary proteins diluted in 0.6 mL of incubation buffer for 3 h at room temperature. After incubation, the microarray was rinsed twice with 1 × PBST (0.2% Tween 20 in 1 × PBS) for 5 min each, and finally rinsed in 1 × PBS before drying. The microarrays were scanned using a Genepix 4000B confocal scanner (Axon Instruments, Foster City, Calif., USA) set at 70% photomultiplier tube and 100% laser power. The acquired images were analyzed at 532 nm for Cy3 detection by Genepix 3.0 software.

Liu X., Yu H., Qiao Y., Yang J., Shu J., Zhang J., Zhang Z., He J, & Li Z. (2018). Salivary Glycopatterns as Potential Biomarkers for Screening of Early-Stage Breast Cancer. EBioMedicine, 28, 70-79.

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Lectin Microarray Analysis of Protein Profiles

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The manufacture of the lectin microarray and data acquisition were performed as described previously [26 (link)–28 (link)]. The proteins isolated from cells or tissue were labeled with Cy3 fluorescent dye (GE Healthcare, Biosciences, Piscataway, NJ, USA) and purified using a Sephadex-G25 column (GE Healthcare). Subsequently, 4 μg of labeled protein was applied to the lectin microarrays and incubated in the chamber at 37 °C for 3 h. After washing and centrifugation, the slides were scanned using a confocal scanner (4000B, AXON Instruments, USA). The fluorescence intensities were extracted by GenePix 7.0 software (Axon). After filtration and normalization, the parallel datasets were compared with each other based on fold changes according to the following criteria: fold changes ≥ 1.50 or ≤ 0.67 and p < 0.05 indicated upregulation or downregulation, respectively. Significant differences in lectin between samples were evaluated using Student’s t test.

Yu H., Li M., Wen X., Yang J., Liang X., Li X., Bao X., Shu J., Ren X., Chen W., Li Z, & Li Y. (2022). Elevation of α-1,3 fucosylation promotes the binding ability of TNFR1 to TNF-α and contributes to osteoarthritic cartilage destruction and apoptosis. Arthritis Research & Therapy, 24, 93.

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Brain Protein Extraction and Labeling

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The animals’ brains were cut into pieces and transferred to a homogenizer. The precooling tissue lysis buffer was rapidly added to the homogenizer and the mixture was fully ground under ice bath conditions. Then the grinding fluid was transferred to a centrifuge tube (2 mL) and subjected to centrifugation (4 °C, 12,000 rpm, 15 min). After centrifugation, the supernatant, which is the tissue protein extract, was transferred to a new centrifuge tube (1.5 mL). The brain proteins were labeled with Cy3 fluorescent dye (GE Healthcare, Buckinghamshire, UK) and purified using Sephadex G-25 columns according to the instructions provided by the manufacturer. Subsequently, the Cy3-labeled brain proteins were quantified and stored at −20 °C in the dark until use.

Cuan Y., He X., Zhao Y., Yang J., Bai Y., Sun Y., Zhang Q., Zhao Z., Wei X, & Zheng X. (2017). Anticonvulsant Activity of Halogen-Substituted Cinnamic Acid Derivatives and Their Effects on Glycosylation of PTZ-Induced Chronic Epilepsy in Mice. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 23(1), 76.

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Labeling and Purification of Lectins and Viruses

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Maackia amurensis lectin II (MAL-II) and sambucus nigra agglutinin (SNA) (Vector, Burlingame, CA) were labelled with Cy5 fluorescent dye and Cy3 fluorescent dye (GE Healthcare, Buckinghamshire, UK), respectively, and the H5N1 subtype strain and H1N1 vaccine were labelled with Cy5 fluorescent dye [34 (link)]. All labelled lectins and viruses were purified using Sephadex G-25 columns, as described previously [35 (link)]. Subsequently, the Cy3- and Cy5-labelled lectins and Cy5-labelled viruses were quantified and stored at −20 °C in dark until use.

Yang J., Li H., Jia L., Lan X., Zhao Y., Bian H, & Li Z. (2019). High expression levels of influenza virus receptors in airway of the HBV-transgenic mice. Epidemiology and Infection, 147, e297.

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Labeling and Purification of BALF Proteins

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First, 100 μg of BALF proteins were labeled with Cy3 fluorescent dye (GE Healthcare, Buckinghamshire, UK). Next, labelled proteins were separated from the excess free dye by Sephadex G-25 columns (GE Healthcare) according to the manufacturer’s instructions. Finally, the purified Cy3-labeled BALF proteins were quantified and stored at -20°C in the dark until processing.

Liu L., Li D., Shu J., Wang L., Zhang F., Zhang C., Yu H., Chen M., Li Z, & Guo X. (2021). Protein Glycopatterns in Bronchoalveolar Lavage Fluid as Novel Potential Biomarkers for Diagnosis of Lung Cancer. Frontiers in Oncology, 10, 568433.

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Salivary Protein Labeling and Preparation

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The collection protocol has been described in previous literature [19] . All donors were asked to avoid eating, drinking, smoking, or using oral hygiene products for at least 1 h before sample collection. The whole saliva (about 1 mL) was collected and placed on ice and the Protease Cocktail Inhibitor (Sigma Aldrich, United States) was added to the saliva immediately after collection. The whole saliva was then centrifuged at 10,000 g for 15 min at 4 °C. The supernatant was collected and stored at -80 °C. Before incubation, the salivary proteins were labeled with Cy3 fluorescent dye (GE Healthcare, Buckinghamshire, England) and purified using Sephadex G-25 columns. Subsequently, the Cy3-labeled salivary proteins were quantified and stored at -20 °C in the dark until use.

Tang Z., Zhang F., Wang Y., Zhang C., Li X., Yin M., Shu J., Yu H., Liu X., Guo Y, & Li Z. (2022). Diagnosis of hepatocellular carcinoma based on salivary protein glycopatterns and machine learning algorithms. Clinical chemistry and laboratory medicine, 60(12).

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