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Alexa fluor 647 n hydroxysuccinimide nhs ester

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Alexa Fluor 647 N-hydroxysuccinimide (NHS) ester is a fluorescent labeling reagent. It is used to covalently attach the Alexa Fluor 647 dye to primary amines on proteins and other biomolecules.

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Lab products found in correlation

Amicon ultra centrifugal filter, by Merck Group (1 mentions) Confocal microscopy, by Leica (1 mentions) Countess 2 cell counter, by Thermo Fisher Scientific (1 mentions) Accuri c6 flow cytometer, by BD (1 mentions) Ack lysing buffer, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Alexa Fluor 647 (1520 citations) Alexa 647 (424 citations) Alexa Fluor 647 NHS Ester (144 citations) N-hydroxysuccinimide (86 citations) N-hydroxysuccinimide (NHS) (78 citations) Alexa Fluors (21 citations) Alexa Fluor 647-NHS (19 citations) Alexa 647-NHS (10 citations) NHS-esters (2 citations)

4 protocols using alexa fluor 647 n hydroxysuccinimide nhs ester

1

Labeling and Analyzing Adenovirus Particles

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Purified HAdV55ΔE1ΔE3 virions were labelled with Alexa Fluor 647 N-hydroxysuccinimide (NHS) ester (Thermo Fisher Scientific) according to manufacturer’s guidelines. In brief, 1 × 1011 v.p. of HAdV55ΔE1ΔE3 was added with 0.2 M Alexa Fluor 647 NHS ester in PBS at 4°C for 2 h. After coupling, unbound dye was removed by separation on Amicon Ultra Centrifugal Filters (Millipore). For confirmation, the labelled and unlabelled virions were separately added onto confluent A549 cells (1 × 104 v.p. per cell). After incubation for 2 h, cells (ratios of cells infected by labelled virions versus those by unlabelled virions: 100: 0, 70: 30, 30: 70, 0: 100, respectively) were washed and analysed by Accur C6 Plus Flow Cytometer (BD Biosciences).
Liu X., Li Z., Li X., Zhang X., Zheng Y., Su W., Feng Y., Liu Y., Wu W., Sun X., Wang N., Ye X., Zhou Z., Liu W., He J., Wang W., Qu L., Zhou R., Chen L, & Feng L. (2024). Neutralizing monoclonal antibodies protect against human adenovirus type 55 infection in transgenic mice and tree shrews. Emerging Microbes & Infections, 13(1), 2307513.
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2

Labeling Lung Vasculature in Mice

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NGs were synthesized as described above with 70 kDa rhodamine-B isothiocyanate conjugated dextran (Sigma), rather than 70 kDa dextran. Alexa Fluor 647 N-hydroxysuccinimide (NHS) ester (ThermoFisher) was conjugated to PECAM antibody (clone MEC13.3) via incubation of 10-fold molar excess of dye with antibody for 30 min at room temperature in 0.1 M sodium bicarbonate in phosphate-buffered saline (PBS), followed by removal of excess dye via five filtrations against 10 kDa molecular weight cutoff centrifugal filter. PLVAP-targeted rhodamine-NGs were administered to C-57BL/6 mice via intravenous bolus. Twenty min after NG injection, Alexa Fluor 647-conjugated PECAM antibody was administered via intravenous bolus. Mice were sacrificed 30 min after NG injection, and lungs were excised, rinsed with PBS, suspended in OCT, and flash frozen in liquid nitrogen. Frozen lungs were sectioned in 10 or 20 μm slices and mounted on slides with DAPI mounting media. Fluorescence images detecting DAPI, rhodamine, and Alexa Fluor 647 were obtained via confocal microscopy (Leica), and images were analyzed in ImageJ.
Myerson J.W., McPherson O., DeFrates K.G., Towslee J.H., Marcos-Contreras O.A., Shuvaev V.V., Braender B., Composto R.J., Muzykantov V.R, & Eckmann D.M. (2019). Cross-linker-Modulated Nanogel Flexibility Correlates with Tunable Targeting to a Sterically Impeded Endothelial Marker. ACS nano, 13(10), 11409-11421.
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3

Phagocytosis Assay with Fluorescent Bacteria

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After assays and cellular detachment, trypan blue was added to cells following enumeration on a Countess II cell counter (Life Technologies). Phagocytosis assays were conducted at an MOI of 1 with Alexa Fluor 647 N-hydroxysuccinimide (NHS) ester (Invitrogen)-labeled strains for 5 min and 30 min at 37°C. Cells were then washed 3 times and detached with TrypLE Express. Cells were mixed with PBS and analyzed using an Accuri C6 flow cytometer (BD Biosciences).
Kim G.L., Hooven T.A., Norambuena J., Li B., Boyd J.M., Yang J.H, & Parker D. (2021). Growth and Stress Tolerance Comprise Independent Metabolic Strategies Critical for Staphylococcus aureus Infection. mBio, 12(3), e00814-21.
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4

Fluorescent Labeling and Biodistribution of IL-10 Constructs

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WT IL-10 and Fab-IL-10 constructs were fluorescently labeled using Alexa Fluor 647 N-hydroxysuccinimide (NHS) ester (Invitrogen), and unreacted dye was removed with a Zeba Spin Desalting Column (7K MWCO, Thermo Scientific) according to the manufacturer’s instruction. WT IL-10 and Fab-IL-10 (5 μg per mouse) were injected intravenously through the tail vein. After 2 hours, the blood was collected, and the spleen and aorta were harvested following perfusion and euthanasia. Red blood cells were lysed following 3 × 3 min incubations with ACK Lysing Buffer (Gibco). The remaining white blood cells were washed with PBS twice and plated for surface staining, as described above. The spleen and aorta were processed and stained as described above.
Volpatti L.R., de Matos S.N., Borjas G., Reda J., Watkins E.A., Zhou Z., Nguyen M., Solanki A., Fang Y, & Hubbell J.A. (2024). LDL-Binding IL-10 Reduces Vascular Inflammation in Atherosclerotic Mice. bioRxiv.
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