All chemicals were of analytical grade and used as received without further purification. Calcium chloride dihydrate (CaCl2·2H2O), anhydrous sodium carbonate (Na2CO3), glycerol, ethylenediaminetetraacetic acid (EDTA), dextran sulfate sodium (DS) salt (MW 50 kDa), poly-L-arginine hydrochloride (Parg) with MW 15–70 kDa, sodium chloride (NaCl), phosphate buffered saline (PBS), isopropyl-β-D-1-thiogalactopyranoside, Rhodamine 6G (MW 479), Fluorescein isothiocyanate (FITC-) dextran (MW 40 kDa), doxorubicin hydrochloride (MW 580) and Hoechst 33258 were purchased from Sigma-Aldrich; DMSO (dimethyl sulfoxide, 99.5%), Trypan Blue solution, phosphate-buffered saline (PBS, pH 7.4), 0.25% (v/v) trypsin-EDTA solution, Versene solution, Dulbecco’s modified Eagle’s medium with Phenol Red (DMEM) and MTT (Thiazolyl Blue Tetrazolium Bromide, 98%) were purchased from PanEko (Moscow, Russia). Fetal bovine serum (FBS) was obtained from PAA Laboratories (Pasching, Austria). Fluorescent dyes sulfo-Cyanine3 maleimide and BDP FL maleimide were obtained from Lumiprobe (Moscow, Russia). Deionized water from a three-stage Milli-Q Plus purification system was used in the experiments.
Sulfo cyanine3 maleimide
Manufactured by Lumiprobe
Sourced in United States
Sulfo-Cyanine3 maleimide is a fluorescent dye that can be used to label and detect biomolecules. It contains a maleimide functional group, which allows it to form covalent bonds with thiol-containing compounds, such as proteins and peptides.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 nhs ester, by Thermo Fisher Scientific (2 mentions)
Fluorescein isothiocyanate fitc dextran, by Merck Group (1 mentions)
Anhydrous sodium carbonate, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Glycerol, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (1 mentions)
Ethylenediaminetetraacetic acid, by Merck Group (1 mentions)
Rhodamine 6g, by Merck Group (1 mentions)
Doxorubicin hydrochloride, by Merck Group (1 mentions)
Calcium chloride dihydrate, by Merck Group (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Poly l arginine hydrochloride, by Merck Group (1 mentions)
Desalting column, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 nhs ester, by Thermo Fisher Scientific (1 mentions)
Alamarblue reagent, by Thermo Fisher Scientific (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Wst 1 reagent, by Merck Group (1 mentions)
Pan caspase inhibitor z vad fmk, by Santa Cruz Biotechnology (1 mentions)
Cc mount tissue mounting medium, by Merck Group (1 mentions)
7 protocols using sulfo cyanine3 maleimide
1
Synthesis and Characterization of Nanoparticles
2
Fluorescent Protein Labeling Protocol
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Sulfo-cyanine3 maleimide (Lumiprobe) was dissolved in dimethyl sulfoxide and incubated with TRL-1 proteins for 1 h at room temperature (fluorophore-to-protein molar ratio was 1:100). Buffer containing 20 mM Hepes pH 7.5, 500 mM NaCl was added to quench the reaction. The fluorophores and other small molecules were removed from the reaction through a desalting column (Thermo Fisher, 89882).
3
Fluorescent Protein Labeling Protocol
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Alexa Fluor 488 NHS ester (Thermo Fisher Scientific), Sulfo-Cyanine3 maleimide (Lumiprobe), and Alexa Fluor 647 NHS ester (Thermo Fisher Scientific) were dissolved in DMSO and incubated with the corresponding protein at room temperature for 1 h (fluorophore to protein molar ratio was 1:1). The reaction was quenched by 20 mM HEPES, pH 7.4, and 500 mM NaCl. The fluorophores and other small molecules were removed from the proteins by passing the reaction mixture through a desalting column (89882; Thermo Fisher Scientific) with buffer containing 20 mM HEPES, pH 7.4, 500 mM NaCl. In imaging assays, fluorophore-labeled proteins were further diluted with the corresponding unlabeled proteins in the same buffer.
4
Fluorescent Protein Labeling Protocol
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Alexa Fluor-488 NHS ester (green; Thermo Fisher Scientific) and Sulfo-Cyanine3 maleimide (red; Lumiprobe) were dissolved in DMSO and incubated with the corresponding protein at room temperature for 1 h (fluorophore to protein molar ratio was 1:1). The reaction was quenched by 20 mM Hepes, pH 7.4, and 1 M NaCl. The fluorophores and other small molecules were removed from the proteins by passing the reaction mixture through a desalting column (17–0851-01; GE Healthcare) with buffer containing 20 mM Hepes, pH 7.4, and 1 M NaCl. In imaging assays, fluorescence-labeled proteins were further diluted with the corresponding unlabeled proteins in the same buffer.
5
Fluorescent Labeling of DR5-B-iRGD Protein
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To obtain fluorescently labeled protein DR5-B-iRGD, first the DR5-B-iRGD amino acid sequence was genetically modified at the N-end by replacing the amino acid residue valine in the 114 position to cysteine by site-directed mutagenesis. The cysteine-modified proteins DR5-B and DR5-B-iRGD were obtained by the method earlier reported for DR5-B [45 (link)]. Further, the cysteine-modified proteins DR5-B and DR5-B-iRGD were labeled by maleimide chemistry coupling with the fluorescent dye sulfo-Cyanine 3 maleimide (Lumiprobe, Moscow, Russia) according to the manufacturer’s protocol.
6
Optimizing Glioblastoma Cell Assays
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The ampicillin, IPTG (isopropyl-β-d-1-thiogalactopyranoside), WST-1 reagent, and CC/Mount tissue-mounting medium were obtained from Sigma-Aldrich (St. Louis, MO, USA); the Alamar Blue reagent was from Thermo Fisher Scientific (Waltham, MD, USA); the iRGD peptide was from InvivoChem (Libertyville, IL, USA); sulfo-Cyanine 3 maleimide was from Lumiprobe (Moscow, Russia); pan-caspase inhibitor Z-VAD-FMK was from Santa Cruz Biotechnology (Dallas, TX, United States). All other chemicals were obtained from Applichem (Darmstadt, Germany) unless otherwise specified. All solvents and components of buffer solutions were of analytical grade. Monoclonal antibodies to TRAIL (MAB375) were from R&D systems (Minneapolis, MN, USA); monoclonal antibodies to DR5 (DR5-01-1) and integrin αVβ3 (23C6), secondary antibodies Dylight 488 and mouse IgG1 (15H6) were from GeneTex (Irvine, CA, USA). E. coli SHuffle B T7 cells were from New England Biolabs (Ipswich, MA, USA. Bacterial cells were cultivated using Gibco Bacto yeast extract and Gibco Bacto tryptone (Thermo Fisher Scientific, Waltham, MA, USA). Human glioblastoma U87 and T98G cells were from ATCC (Washington, DC, USA). The cell culture media DMEM, 0.25% Trypsin-Versene solution, and phosphate-buffered saline tablets were from PanEco (Moscow, Russia). Thefetal bovine serum was from HyClone (Cramlington, UK).
7
Synthesis and Conjugation of Barium Titanate Nanoparticles
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