Before conjugation, all antibodies were purified as above. The near-infrared fluorophore Cy5.5-NHS (GE Healthcare) was resuspended in methanol, aliquoted, and dried by SpeedVac. Using a molar excess of 3:1, the antibody was labeled, and the pH was adjusted to 8.5 with Na2CO3. The reaction was shaken at 22°C for 4 hours, followed by gel purification (PD10) and ultrafiltration (Amicon). The number of dye molecules per antibody was evaluated using a spectrophotometer and calculated to be 1.3 (SpectraMax M5, Molecular Devices). The dyelabeled antibody conjugate was freshly prepared for each experiment.
Cy5.5 nhs
Manufactured by GE Healthcare
Sourced in United States
Cy5.5-NHS is a fluorescent dye that can be used to label biomolecules such as proteins, nucleic acids, and other molecules. It has an excitation wavelength of approximately 675 nm and an emission wavelength of approximately 694 nm, making it suitable for detection in the far-red/near-infrared region of the spectrum. The NHS (N-hydroxysuccinimide) ester group allows for covalent attachment of the dye to primary amine groups on target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Spectramax m5, by Molecular Devices (1 mentions)
2 iminothiolane hydrochloride, by Merck Group (1 mentions)
Albumin from bovine serum bsa, by Merck Group (1 mentions)
Nh2 peg5800 b pcl19000, by Polymer Source (1 mentions)
Tris 2 carboxyethyl phosphine tcep, by Thermo Fisher Scientific (1 mentions)
Succinimidyl 3 2 pyridyldithio propionate spdp, by Thermo Fisher Scientific (1 mentions)
Amicon ultra 100k, by Merck Group (1 mentions)
8 protocols using cy5.5 nhs
1
Fluorescent Antibody Labeling Protocol
2
Synthesis and Characterization of Targeted Theranostic Nanoparticles
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All materials were purchased from commercial suppliers and used without further purification, unless otherwise noted. Cy5.5-NHS was purchased from GE Healthcare (Piscataway, NJ, USA). Succinimidyl iodoacetate (SIA), 2-iminothiolane hydrochloride (Traut's reagent), albumin from bovine serum (BSA), and Tf were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methoxy-PEG5000-b-PCL15000 was purchased from Daigang Biomaterial Co., Ltd. (Jinan, Shandong, China). NH2-PEG5800-b-PCL19000 was purchased from Polymer Source Inc. (Montreal, Quebec, Canada). Other chemicals and reagents used in the study were of analytical grade.
All cell lines were preserved in our lab. BALB/c nude mice (male, 4 to 6 weeks) were purchased from Hunan Slake Jingda Experimental Animal Co. Ltd., Changsha, China (Animal Qualification Certification No. 43004700000595). All animal studies were approved by the Animal Experimentation Ethics Committee of College of Life Science and Technology, Huazhong University of Science and Technology and carried out in compliance with the guidelines approved by the Science and Technology Department of Hubei Province.
All cell lines were preserved in our lab. BALB/c nude mice (male, 4 to 6 weeks) were purchased from Hunan Slake Jingda Experimental Animal Co. Ltd., Changsha, China (Animal Qualification Certification No. 43004700000595). All animal studies were approved by the Animal Experimentation Ethics Committee of College of Life Science and Technology, Huazhong University of Science and Technology and carried out in compliance with the guidelines approved by the Science and Technology Department of Hubei Province.
3
Fluorescent Labeling of Ovarian Cancer Cells
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Cy5.5-NHS was purchased from GE Healthcare (Piscataway, NJ, USA). Mouse Anti-Human CD227 antibody (cat. no. 6378-0150, 0108) was purchased from RayBiotech, Inc. (Norcross, GA, USA). Mouse IgG (cat. no. A7208) and fluorescein-NHS were purchased from Beyotime Institute of Biotechnology (Haimen, China). Other chemicals were of analytical grade or better and were used as purchased from Sigma-Aldrich, St. Louis, MO, USA. The human ovarian carcinoma OVCAR3 cell line was purchased from Shanghai Cell Bank of the Chinese Academy of Science (Shanghai, China). Ethics approval was provided by Wenzhou Medical University (Wydw2014-0134).
4
Monitoring UCNP-Loaded Hb μGel Injection
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The injection site of UCNP-loaded Hb μGels was monitored by computerized tomography (CT; SKY-scan 1076, Broker, USA) at 0, 1, and 7 days. Cy5.5-Hb μGel, which was modified with Cy5.5-NHS (GE Healthcare, Chicago, USA), was injected directly into the tumors of 4T1 cell-xenografted mice, and fluorescence signals were visualized at predetermined times and at wavelengths corresponding to Cy5.5 and hemoglobin using FOBI in vivo imaging equipment (NeoScience, Suwon, R. O. Korea). Separately, the major organs (liver, kidney, spleen, lung and heart) of the mouse were exicsed and visualized at 24 h after sacrifice of mice.
5
Magnetic Nanoparticle-Mediated miRNA Delivery
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The two microRNA mimics were provided by Integrated DNA Technology (IDT, Coralville, IA). The sequence of miR-4539 is 5′-S-S-GCUGAACUGGGCUGAGCUGGGC and the sequence of miR-4261 is 5′-S-S-AGGAAACAGGGACCCA. The 5′-end of the oligo was modified with disulfide capping to introduce a thiol group for conjugation with magnetic nanoparticles. The culture media, succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and tris(2-carboxyethyl)phosphine (TCEP) were purchased from Thermo Fisher Scientific (Waltham, MA), while the fluorescent dye Cy5.5-NHS was purchased from GE Healthcare Life Sciences (Pittsburgh, PA). All antibodies for Western blotting were purchased from Abcam (Cambridge, MA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
6
Synthesis and Characterization of Cy5.5-iRGDC-BK01
7
Labeling Protein Nanoparticles for In Vivo Imaging
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For in vivo imaging of LNs, NIR dye, Cy5.5 was used to label the five different protein nanoparticles (DPS, PTS, HBVC, hFTN, and hFTN-RFP) and RFP. 2 mmol of Cy5.5 N-hydroxysuccinimide (NHS) ester (Cy5.5-NHS, GE Healthcare, Piscataway, NJ) with excitation and emission maximum wavelength of 675 and 693 nm, respectively, was incubated with the purified protein nanoparticles in sodium bicarbonate (0.1 M, pH 8.5) at room temperature for 12 h. Cy5.5-labeled protein nanoparticles were loaded onto a sucrose step gradient (40, 35, 30, 25, and 20%w/v) and centrifuged at 35,000 rpm for 16 h at 4 °C to separate the unbound Cy5.5 from Cy5.5-labeled protein nanoparticles or RFP. Subsequently, sucrose solution (20–25% sucrose) containing the Cy5.5-labeled protein nanoparticles or RFP was fractionated and then exchanged to PBS (2.7 mM KCl, 137 mM NaCl, 2 mM KH2PO4, 10 mM Na2HPO4, pH 7.4) by ultrafiltration (Amicon Ultra 100K, Millipore, Billerica, MA).
8
In situ PEG-Hydrogel for Tumor Imaging
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The in situ gelling PEG-hydrogel loaded with o-NBA/Cy5.5-HAase-HSA-NPs (100 μl) were directly injected to the tumor of the AsPC-1 cell-xenografted mice. Separately, an aliquot (100 μl) of PTX-Cy5.5-HSA-NPs was injected via the tail vein of AsPC-1 cell-bearing mice. The HAase and HSA had been modified with Cy5.5-NHS (GE Healthcare, Chicago, USA) and dialyzed to remove unreacted Cy5.5 before preparation of HSA nanoparticles. The Cy5.5-fluorescence signals from the tumors were visualized at predetermined times using the FOBI in vivo imaging system (NeoScience, Suwon, Korea).
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