All chemicals and cell culture supplies were obtained from Fisher Scientific (Pittsburgh, PA) and used as received unless stated otherwise. The internal standard solution and multi-element tuning solution for ICP-MS were purchased from VHG Labs (Manchester, NH). All antibodies were purchased from Ab cam (Cambridge, MA) or Santa Cruz Biotechnology (Santa Cruz, CA) and used as received. Hyaluronan was purchased from Lifecore Biomedical (Chaska, MN). The poly-L-Lysine coated glass coverslips were purchased from BD Biosciences (Franklin Lakes, NJ). The Cy7 N-hydroxysuccinimide (NHS) ester was purchased from Lumiprobe (Hallandale Beach, FL). The Lysotracker blue™ was purchased from Invitrogen. The human oral squamous carcinoma cell line, MDA-1986, was a gift from Dr. Jeffrey Myers (University of Texas, M.D. Anderson Cancer Center; Houston, TX).
Cy7 n hydroxysuccinimide nhs ester
Manufactured by Lumiprobe
Sourced in United States
Cy7 N-hydroxysuccinimide (NHS) ester is a fluorescent dye molecule commonly used in bioconjugation reactions. It is a compact and water-soluble ester that can form covalent bonds with primary amine groups, allowing it to be attached to various biomolecules such as proteins, peptides, or nucleic acids.
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2 protocols using cy7 n hydroxysuccinimide nhs ester
1
Characterization of MDA-1986 Oral Carcinoma Cells
2
Cy7 Conjugation of Antibodies
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Conjugation of Cy7 dye with COC183B2 or a mouse isotype IgG antibody was performed according to the manufacturers’ instructions. Briefly, 1 mg of Cy7 N-hydroxysuccinimide (NHS) ester (Lumiprobe, Hunt Valley, MD, USA) was dissolved in 147 μL of dimethyl sulfoxide (DMSO) and added to an antibody solution to keep the final concentration of DMSO below 5%. Antibodies (1 mg, 2.8 μmol/L) were incubated with Cy7 NHS ester (56 μmol/L) (mole ratio=1:20) in boric acid/NaOH (pH: 8.3−8.5) at 4 °C protected from light overnight. The eluted solution contained conjugated products, and excess unbound dye was removed using a Zeba Spin Desalting Column, 0.5 mL, 7K MWCO (Thermo Fisher Scientific, Rockford, IL, USA). The protein concentrations of Cy7-conjugated samples were determined by measuring the absorption at 280 nm with a UV-Vis system (Thermo Fisher Scientific). The Cy7 dye concentration was measured as the absorption at 750 nm with the UV-Vis system to determine the number of fluorophore molecules conjugated to each antibody molecule.
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