A total of 25 mg Alexa Fluor 750 NHS Ester (Invitrogen) was dissolved in 45 μl of 0.4 M NH2-LPETGG peptide in DMSO and incubated at room temperature for 6 h. A total of 2.5 μl DIPEA was then added and incubated at room temperature overnight. Reactions were quenched by the addition of 450 μl of 1 M Tris, pH 7.5, which were incubated on ice for 2 h. The reaction product was purified on a preparative Kromasil 100-5-C18 column (21.2 × 250 mm, Peeke Scientific) by reverse phase HPLC (flow rate: 9.5 ml min−1; gradient: 10–70% acetonitrile with 0.1% TFA in 0.1% aqueous TFA gradient over 30 min; retention time 8 min) before pooling and lyophilizing the collected fractions. The concentration of the peptide was determined by the known molar extinction coefficient of Alexa Fluor 750, ɛ749nm=290,000 M−1 cm−1.
Alexa fluor 750 nhs ester
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 750 NHS ester is a fluorescent dye used for labeling proteins and other biomolecules. It has an excitation maximum at 749 nm and an emission maximum at 775 nm, making it suitable for applications that require far-red or near-infrared fluorescence detection.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Ivis spectrum ct imaging system, by PerkinElmer (3 mentions)
Zeba column, by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (2 mentions)
Nap 5 size exclusion column, by GE Healthcare (2 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Methanol, by Thermo Fisher Scientific (2 mentions)
Lsrii facs analyzer, by BD (1 mentions)
Trustain fc receptor block, by BioLegend (1 mentions)
Nbd pe, by Thermo Fisher Scientific (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Cholesterol, by Merck Group (1 mentions)
Dspe peg2000, by Avanti Polar Lipids (1 mentions)
3 aminopropyltriethoxysilane aptes, by Merck Group (1 mentions)
Dbco nhs ester, by Vector Laboratories (1 mentions)
Dulbecco s modified eagle medium dmem growth medium, by Thermo Fisher Scientific (1 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
18 protocols using alexa fluor 750 nhs ester
1
Alexa Fluor 750 Peptide Conjugation
2
Isolation and Quantification of CNS-Infiltrating Immune Cells in EAE
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CNS-infiltrating immune cells from EAE animals during the inflammatory phase 12–17 d after immunization were isolated and quantified as we described previously (Ufer et al., 2016 (link)). We stained single-cell suspensions in the presence of TruStain Fc receptor block (BioLegend) and used Alexa Fluor 750 NHS Ester (Invitrogen) for live/dead discrimination. The antibodies and the respective antigen, host species, supplier, catalog number, clone, and dilution are listed in Table S8 . Data were acquired on an LSR II FACS analyzer (BD Biosciences).
3
Biodistribution of Alexa750-ApoE NTD-scFv
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ApoE NTD-scFv was labeled with Alexa Fluor™ 750 NHS ester (Invitrogen) and used for preparing the complex with DMPC, which was sterilized by filtering through 0.22 µm-diameter pores. The body distribution of Alexa750-ApoE NTD-scFv + DMPC was measured as described previously. 2 Briefly, Alexa750-ApoE NTD-scFv + DMPC (Alexa750-ApoE NTD-scFv: 12.5 µM; DMPC: 0.8 mg mL -1 ) was injected into anesthetized mice (C57Bl/6N; male; 9-10 weeks old; 22.7-24.8 g; Japan SLC, Japan) intravenously (for the Alexa750-ApoE NTD-scFv concentration of approximately 1 µM in the blood). After 4 h-long incubation, the mice underwent thoracotomy and laparotomy. Urine and blood were collected, and PBS containing 10 U mL -1 heparin was perfused through the entire body. The organs (the heart, lungs, liver, spleen, kidneys, and intestines) were excised, and plasma was prepared from heparinized blood by centrifugation. Each excised organ was homogenized, and the fluorescence at 776 nm of the resultant suspension (excitation at 749 nm) was measured using a plate reader. Three animals were used in this study (n = 3).
4
Nanoparticle Formulation and Characterization
5
In Vivo Tracking of Tumor Antigen
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SA-PD-L1 was labeled with Alexa Fluor 750 NHS Ester (Thermo Fisher Scientific) as described (19 (link)). Labeled protein (1 μg) was tethered to 1000 microgels. Microgels displaying SA-PD-L1 or free labeled protein were transplanted in the EFP of nondiabetic BALB/c recipients (1200 microgels per EFP, n = 4 SA-PD-L1–presenting microgels, n = 5 free protein). Signal intensity was monitored over 20 days using an IVIS SpectrumCT imaging system (PerkinElmer). Intensity measurements were normalized to day 0 values.
6
Functionalization of Silica Surfaces with APTES
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3-Aminopropyl-triethoxysilane (APTES) was obtained from Sigma-Aldrich, USA. The nanocavity chips were cleaned with acetone, methanol, and isopropanol prior to binding experiments. A 1% APTES solution was prepared in toluene (anhydrous) and allowed to bind on the chips for 30 min. This step functionalizes the silica surface with amino groups available for further binding. The substrates were washed with toluene to remove weakly bound APTES molecules. The chips were baked at 110 °C for 30 min and then cleaned again with DI water for 15 min. The samples were then dried with nitrogen. Alexa Fluor 750 NHS ester was purchased from Thermofisher Scientific USA and dissolved in DMSO (1 mg/ml). The solution was added to the substrates with stirring and dye molecules were allowed to bind to the functionalized substrates for one hour. The chips were then cleaned well with DMSO and water. A drop of water was placed on the chips, covered with a coverslip, and then imaged.
7
Synthesis and Labeling of Peptides
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All peptides were synthesized using Fmoc solid-phase chemistry on a CEM Liberty Blue microwave-assisted synthesizer and purified with high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization–mass spectrometry (MALDI-MS). Biotinylated peptides were synthesized on-resin by reacting Biotin-ONp (33755-53-2, Novabiochem) with amine-terminated peptides in a threefold excess overnight in N,N′-dimethylformamide (DMF). DBCO was conjugated with peptides by reacting DBCO-NHS Ester (A133-100, Click Chemistry Tools) with amine-terminated peptide on-resin overnight in DMF in the presence of diisopropylethylamine (DIEA). TAMRA-labeled peptides were prepared on-resin by reacting 5(6)-TAMRA (AS-81124, Anaspec Inc.) in a threefold excess with the addition of N,N′-diisopropylcarbodiimide (DIC) and 6-chloro-1-hydroxybenzotriazole (HOBt-Cl). Alexa Fluor 750–labeled peptides were prepared on-resin by reacting Alexa Fluor 750 NHS Ester (A37575, Thermo Fisher Scientific) with amine-terminated peptides in the presence of DIEA overnight in DMF.
8
In Vivo Monitoring of Immobilized SA-FasL
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SA-FasL was labelled with AlexaFluor750 NHS Ester (Thermo Fisher), and free dye was removed by desalting in Zeba column (7k MWCO, Thermo Fisher) three times. 3.0 µg of labelled SA-FasL was immobilized onto 2,000 biotin microgels by incubation for 30 min followed by 5 wash steps. Microgels presenting SA-FasL or free SA-FasL were implanted under the kidney capsule of C57Bl/6 recipients (n=8 mice/group), and signal intensity and distribution were monitored longitudinally using an IVIS SpectrumCT imaging system (Perkin-Elmer). Intensity measurements were normalized to day 0 values. Non-linear single exponential decay curve fits were performed in GraphPad Prism and retention time was compared using a t-test. Area-under-the-curve metric was calculated for each group, and a Welch’s t-test was used to compare groups.
9
In Vitro and In Vivo Cancer Nanomedicine
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BSA, MPC, fluorescein isothiocyanate (FITC), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Glycerol dimethacrylate (GDA), ammonium persulfate (APS), N,N,N',N'-tetramethylethylenediamine (TEMED) and succinic anhydride (SA) were purchased from Aladdin Industrial (Shanghai, China). N-(3-Aminopropyl) methacrylamide hydrochloride (Apm) was purchased from Polymer Science. N-Succinimidyl 4-forMylbenzoate (SFB) and Dox were purchased from J&K Chemical (Shanghai, China). MA-BzA was synthesized by mixing Apm and SFB under stirring in DMSO for 2 h. Alexa Fluor® 750 NHS Ester was purchased from Thermo Fisher Scientific. All the chemicals were commercially available and used as without further purification. HepG2 cells were purchased from American Type Culture Collection (ATCC). Fetal bovine serum (FBS) was purchased from Lonza Walkerrsville. The Dulbecco's Modified Eagle Medium (DMEM) growth medium and penicillin/streptomycin were purchased from Invitrogen. Female Balb/C nude mice (6 weeks old) were purchased from Guangdong Province Medical Animal Center and maintained in an SPF (specific pathogen free) class experimental animal room.
10
In Vivo Monitoring of Immobilized SA-FasL
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