Alexa fluor 647 nhs ester

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

Alexa Fluor 647 NHS Ester is a fluorescent dye used for labeling and detection in various biological applications. It has an absorption maximum at 650 nm and an emission maximum at 665 nm, making it suitable for detection in the red/far-red region of the visible spectrum.

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Alexa Fluor 647 NHS Succinimidyl Ester (2 citations) Alexa Fluor 647 NHS ester dye (2 citations) Alexa Fluor 647 NHS ester (AF647, (2 citations) Alexa Fluor 647 N-hydroxysuccinimide (NHS) ester (4 citations) Alexa Fluor 647-NHS (19 citations) Alexa 647-NHS (10 citations) Alexa Fluor 647 (1520 citations) NHS-esters (2 citations) Alexa 647 (424 citations) Alexa Fluors (21 citations)

144 protocols using alexa fluor 647 nhs ester

Isolation and Labeling of Anabaena Gas Vesicles

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Native gas vesicles (GVs) were isolated from Anabaena flos-aquae as previously described.^{20 (link)} Concentrations were measured by optical density (OD) at 500 nm using a spectrophotometer (NanoDrop ND-1000, Thermo Scientific). Stripped GVs were prepared by treatment of native GVs with 6M urea solution followed by two rounds of centrifugally-assisted flotation and removal of the subnatant.^{20 (link)} Fluorescently-labeled gas vesicles were prepared by mixing GVs at OD 10 in 1x phosphate-buffered saline (PBS) with 6 μM Alexa Fluor 647 NHS Ester (Invitrogen, prepared as 10 mM solution in dimethyl sulfoxide). Dually-labeled GVs were prepared by mixing GVs at OD10 with 6 μM pHrodo Red succinimidyl ester (Invitrogen, prepared as 10 mM solution in dimethyl sulfoxide) and 18 μM Alexa Fluor 647 NHS Ester (Invitrogen, prepared as 10 mM solution in dimethyl sulfoxide). After rotating in the dark at 25°C for 1 h, the reactions were quenched with Tris-HCl. Prior to use, all GVs were buffer exchanged into 1x PBS by two rounds of overnight dialysis through a regenerated cellulose membrane (12–14 kD MWCO, Repligen).

Ling B., Lee J., Maresca D., Lee-Gosselin A., Malounda D., Swift M.B, & Shapiro M.G. (2020). Biomolecular Ultrasound Imaging of Phagolysosomal Function. ACS nano, 14(9), 12210-12221.

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Vascular and Cytoskeletal Staining

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Tissues were fixed with 4% paraformaldehyde (PFA), frozen in OCT (Tissuetek) and sliced at 10 µm. Human vessels were stained for plasma membrane with the human specific Ulex europaeus agglutinin (UEA) lectin coupled to rhodamine (Vector Laboratories) and for F-actin with phalloidin coupled to AlexaFluor-568 (Invitrogen). Type IV pili were detected with an in-house generated anti-PilE nanobody coupled to AlexaFluor-647 (AlexaFluor-647-NHS-ester, Invitrogen).

Charles-Orszag A., Tsai F.C., Bonazzi D., Manriquez V., Sachse M., Mallet A., Salles A., Melican K., Staneva R., Bertin A., Millien C., Goussard S., Lafaye P., Shorte S., Piel M., Krijnse-Locker J., Brochard-Wyart F., Bassereau P, & Duménil G. (2018). Adhesion to nanofibers drives cell membrane remodeling through one-dimensional wetting. Nature Communications, 9, 4450.

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Readout Probe Design for seqFISH+

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Readout probes of 12–15-nt in length were designed for seqFISH as previously described^{20 (link),21 (link)}. In brief, a set of probe sequences was randomly generated with combinations of A, T, G or C nucleotides with a GC-content range of 40–60%. To minimize cross-hybridization between the readout probes, any probes with ten or more contiguously matching sequences between the readout probes were removed. The readout probes for sequential immunofluorescence were similarly designed except ‘C’ nucleotide is omitted^{60 (link)}. The 5’ amine-modified DNA oligonucleotides (Integrated DNA Technologies) with the readout probe sequences were conjugated in-house to Alexa Fluor 647-NHS ester (Invitrogen A20006) or Cy3B-NHS ester (GE Healthcare PA63101) or Alexa Fluor 488-NHS (Invitrogen A20000) as described before^{20 (link),21 (link)}, or fluorophore conjugated DNA oligonucleotides were purchased from Integrated DNA Technologies. In total, 240 unique readout probes^{21 (link)} were designed and synthesized for DNA seqFISH+ experiments, and subsets of those readout probes were used for RNA seqFISH experiments. The cost for 240 readout probes for DNA seqFISH+ were approximately $15,000 with 5’ amine-modified DNA oligonucleotides and dye conjugation in-house, and $50,000 with fully labeled purchase, which can be used over hundreds or thousands of experiments.

Takei Y., Yun J., Zheng S., Ollikainen N., Pierson N., White J., Shah S., Thomassie J., Suo S., Eng C.H., Guttman M., Yuan G.C, & Cai L. (2021). Integrated spatial genomics reveals global architecture of single nuclei. Nature, 590(7845), 344-350.

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Multiplexed Spatial Transcriptomics via DNA seqFISH+

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To implement the two-layer DNA seqFISH+ strategy, 96 unique readout probes were used in each fluorescent channel for a total of 288 unique readout probes for 3 fluorescent channels. The readouts probe sequences were obtained from our previous DNA seqFISH+ studies^{7 (link),8 (link)} as well as additional orthogonal readout probe sequences were generated and validated similarly to those previous studies. The RNA seqFISH+ readout probe sequences were selected from a subset of the two-layer DNA seqFISH+ readout probe sequences. The readout probe sequences (12-15-nt) for sequential immunofluorescence were selected from our previous studies^{7 (link),8 (link)} and further designed and validated with the same criteria for this study. The fluorescently-labeled readout probes (Integrated DNA Technologies) that can bind to the readout sequences on the primary probes or primary antibodies were conjugated in-house to Alexa Fluor 647–NHS ester (Invitrogen A20006), Cy3B–NHS ester (GE Healthcare PA63101), or Alexa Fluor 488–NHS ester (Invitrogen A20000) as described before^{17 (link)} or directly purchased (Integrated DNA Technologies).

Takei Y., Yang Y., White J., Yun J., Prasad M., Ombelets L.J., Schindler S, & Cai L. (2023). High-resolution spatial multi-omics reveals cell-type specific nuclear compartments. bioRxiv.

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Fluorescent Cell Labeling with NHS Ester

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Labeling with NHS ester was performed as described before (Lund et al., 2018). Briefly, cells grown to mid‐exponential phase (OD₆₀₀ ~ 0.5) were collected by centrifugation and growth medium was discarded. Cells were resuspended in PBS containing 8 μg ml⁻¹ Alexa Fluor 647 NHS ester (Invitrogen) and incubated at room temperature for 5 min. Cells were washed by centrifugation and resuspension in PBS.

Bojer M.S., Wacnik K., Kjelgaard P., Gallay C., Bottomley A.L., Cohn M.T., Lindahl G., Frees D., Veening J., Foster S.J, & Ingmer H. (2019). SosA inhibits cell division in Staphylococcus aureus in response to DNA damage. Molecular Microbiology, 112(4), 1116-1130.

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Functionalization of Nanopillar Substrates

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The nanopillar substrates were cleaned with 1 M potassium hydroxide (Sigma-Aldrich, 221473) solution for 15 min at RT. The substrates were washed with nanopure water five times and air-dried. The substrates were then treated with air plasma for 15 min and attached to plastic dishes, which have a hole punched in the bottom, with silicone sealant. The substrates were rinsed with anhydrous methanol (Sigma-Aldrich, 322415) and incubated in 2 mL of a mixture containing anhydrous methanol, glacial acetic acid (Sigma-Aldrich, 695092), and (3-aminopropyl) triethoxysilane (Sigma-Aldrich, A3648) in a v/v/v ratio of 100:5:3 for 30 min. The substrates were washed with anhydrous methanol five times and then with nanopure water three times. Afterward, the substrates were rinsed with 0.1 M sodium bicarbonate (Sigma-Aldrich, S5761) solution (pH 8) and incubated in 0.1 M sodium bicarbonate solution (pH 8) with 2 μM Alexa Fluor 647 NHS Ester (Invitrogen, A37573) for 30 min. The substrates were washed with PBS five times before imaging.

Roy A.R., Zhang W., Jahed Z., Tsai C.T., Cui B, & Moerner W.E. (2021). Exploring Cell Surface–Nanopillar Interactions with 3D Super-Resolution Microscopy. ACS Nano, 16(1), 192-210.

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Protein Labeling Protocols for Immunoassays

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CTLA4Ig (Orencia, Bristol-Meyers Squibb), human IgG (Sigma), and albumin from chicken egg white (Ovalbumin, Sigma) were reconstituted and conjugated to Alexa Fluor 647 NHS ester, Alexa Fluor 488 NHS ester and Alexa Fluor 568 NHS ester (Invitrogen), respectively, following the protocol provided by the manufacturer. Stock solutions for each molecule (10 mg/ml) were prepared by mixing unlabeled and Alexa Fluor-conjugated molecules at a 19:1 ratio in water for injection (USP sterile grade, RMBIO). A cocktail solution (COMBO) containing 10 mg/ml of CTLA4Ig, Human IgG and Ovalbumin was also prepared by mixing unlabeled and Alexa Fluor-conjugated molecules at a 19:1 ratio in water for injection (USP sterile grade, RMBIO). CTLA4Ig was reconstituted and conjugated to Fluorescein-5-Isothiocyanate (FITC, Invitrogen) according to manufacturer instructions.

Capuani S., Hernandez N., Paez-Mayorga J., Dogra P., Wang Z., Cristini V., Chua C.Y., Nichols J.E, & Grattoni A. (2022). Localization of drug biodistribution in a 3D-bioengineered subcutaneous neovascularized microenvironment. Materials Today Bio, 16, 100390.

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Fluorescent Protein Labeling Protocol

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Protein labeling was performed as previously reported (Feric et al., 2016 (link); Ferrolino et al., 2018 (link)). NPM1 protein was labeled with Alexa Fluor 488 NHS Ester (no. A20000; Invitrogen), DCAF13-Sof/Sof-RKA/Nter proteins were labeled with Alexa Fluor 405 NHS Ester (no. A30000; Invitrogen), and UTP23 was labeled with Alexa Fluor 647 NHS Ester (no. A37573; Invitrogen) according to the manufacturer’s protocol. Briefly, labeling dye was introduced into a 50 μM protein solution prepared by dilution with storage buffer (20 mM HEPES, 500 mM NaCl, 1 mM DTT, pH = 7.5) at a concentration ratio of 1:1 and the solution was incubated for 1 h in the dark. Excessive dye was eluted by Amicon Ultra-3K centrifugal filter devices (no. UFC500396; Millipore). In addition, fluorescently labeled NPM1-A488 was mixed with unlabeled NPM1 at a 1:9 ratio in 6 M guanidine-HCl solution and dialyzed with Slide-A-Lyzer Dialysis Cassettes (3.5 K MWCO, no. 66330; Thermo Fisher Scientific) in storage buffer (20 mM HEPES, 500 mM NaCl, 1 mM DTT, pH = 7.5), followed by concentration to 300 μM by Ultra-3K centrifugal filter devices.

Zhou L., Wang S., Hu W., Liu X., Xu L., Tong B., Zhang T., Xue Z., Guo Y., Zhao J., Lu L., Fan H., Qian W., Chen J., Chen W, & Wang L. (2023). T cell proliferation requires ribosomal maturation in nucleolar condensates dependent on DCAF13. The Journal of Cell Biology, 222(10), e202201096.

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S. aureus Labeling with Alexa Fluor 647

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S. aureus grown to mid-exponential phase (OD₆₀₀ ~0.5) were resuspended in PBS containing Alexa Fluor 647 NHS ester (Invitrogen) at 8 μg ml⁻¹ and incubated at room temperature for 5 min. Cells were then washed by centrifugation and resuspension in PBS.

Lund V.A., Wacnik K., Turner R.D., Cotterell B.E., Walther C.G., Fenn S.J., Grein F., Wollman A.J., Leake M.C., Olivier N., Cadby A., Mesnage S., Jones S, & Foster S.J. (2018). Molecular coordination of Staphylococcus aureus cell division. eLife, 7, e32057.

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Fluorescent Fibronectin Labeling and Uptake

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Plasma fibronectin was isolated in the laboratory, according to Früh et al.^[¹⁰⁶^] Fibronectin was labeled using Alexa Fluor 647 NHS Ester (succinimidyl ester) (Invitrogen A20106), resulting in a degree of labeling of 10 (10 molecules of dye per molecule of fibronectin). During coculture, labeled fibronectin was added every 24 hours in the medium at a concentration of 5 mg mL⁻¹. On day 2 and 3 of coculture, only 50% of culture medium was exchanged by fresh medium containing labeled fibronectin.

Fernandes A., Miéville A., Grob F., Yamashita T., Mehl J., Hosseini V., Emmert M.Y., Falk V, & Vogel V. (2022). Endothelial‐Smooth Muscle Cell Interactions in a Shear‐Exposed Intimal Hyperplasia on‐a‐Dish Model to Evaluate Therapeutic Strategies. Advanced Science, 9(28), 2202317.

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