Three microliters of input samples were mixed with 2 μL of 0.1M Na2CO3 buffer, and the mixture was incubated with 0.2 μL of Alexa Fluor 555 NHS ester (10 mg/mL in DMSO; #A37571, Thermofisher Scientific) for 1 h at RT. Excess dyes were removed via washing with Zeba micro spin desalting columns (#87765, Thermofisher Scientific). After two-time washing, 3 μL of dye-labeled samples were loaded on a glass slide and allowed to settle (30 min, RT). The slide was then washed with fPBS and incubated with 10 μL of fixation buffer (4% paraformaldehyde) and washed. For CD63 detection, captured vesicles were labeled (90 min, RT) with biotinylated anti-CD63 antibody (10 μg/mL), washed in fPBS, and incubated (30 min, RT) with FITC-streptavidin secondary reagents (5 μg/mL). For EpCAM detection, vesicles were incubated (90 min, RT) with anti-EpCAM antibody (10 μg/mL), washed in fPBS, and incubated (30 min, RT) with Alexa Fluor 647 anti-mouse antibody (5 μg/mL). After the final washing step, fluorescence images were taken by an inverted microscope (Nikon, Eclipse TE2000S) equipped with an sCMOS camera (Andor, Zyla). Image analyses were performed using ImageJ.
Alexa fluor 555 nhs ester
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 555 NHS Ester is a fluorescent dye compound used in biological research. It is designed to covalently attach to primary amines, allowing for the labeling of proteins, nucleic acids, and other biomolecules. The Alexa Fluor 555 dye has an excitation maximum of 555 nm and an emission maximum of 565 nm, making it suitable for a variety of fluorescence-based applications.
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Lab products found in correlation
Alexa fluor 647 nhs ester, by Thermo Fisher Scientific (6 mentions)
Zeba micro spin desalting column, by Thermo Fisher Scientific (1 mentions)
Eclipse te2000 s, by Nikon (1 mentions)
Pd 10 column, by Cytiva (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Agilent cary 100, by Agilent Technologies (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Proscanarray express, by PerkinElmer (1 mentions)
Alexa fluor 555 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Ab18251, by Abcam (1 mentions)
Ab176753, by Abcam (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
T7816, by Merck Group (1 mentions)
Alexa fluor 488 nhs ester, by Thermo Fisher Scientific (1 mentions)
Amicon ultra 0.5 centrifugal filter unit, by Merck Group (1 mentions)
Zeba 2 ml 7k mwco spin desalting column, by Thermo Fisher Scientific (1 mentions)
23 protocols using alexa fluor 555 nhs ester
1
Fluorescent Labeling of Extracellular Vesicles
2
Fluorescent Labeling of SARS-CoV-2 Proteins
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RBDs (Wuhan, Delta, and Omicron) and S proteins (Wuhan and Omicron) were covalently labeled with Alexa Fluor™ 555. To this end, the proteins were first diluted to final concentration of 2 μM in buffer containing 2/3 PBS and 1/3 0.1 M carbonate buffer with resulting pH of 8.3. Next, to the protein solutions was added Alexa Fluor™ 555 NHS ester (Thermo‐Fischer) with final concentration of 40 μM. Alexa Fluor™ 555 NHS stock solution was prepared immediately before use by solubilizing the dye in DMSO (Sigma‐Aldrich, >99.9%) to stock concentration of 1.6 mM. The mixtures of the amine‐reactive Alexa Fluor™ 555 NHS ester with the proteins were incubated in dark with continuous shaking for 1 hr at room temperature. After, protein conjugates were separated from unconjugated fluorochrome by using PD‐10 columns (Cytiva) pre‐equilibrated with PBS. For estimation of efficacy of coupling of fluorochrome to RBDs and S proteins, the UV‐visual absorbance spectra of conjugated proteins were recorded in the range 200–700 nm using Agilent Cary 100 absorbance spectrophotometer (Agilent Technologies, Santa Clara, CA).
3
Serum Protein Labeling and Quantification
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Total protein
concentrations of serum samples were measured with a DC protein assay
kit (Bio-Rad Laboratories, Hercules, CA, USA). Each volunteer serum
sample was fluorescently labeled with Alexa Fluor 555 NHS ester (Thermo
Fisher Scientific, Waltham, MA, USA). First, 10 μg of total
protein was diluted in PBS to 27 μL. The pH of the solution
was adjusted with 3 μL of 1 M sodium bicarbonate. Then 0.21
μL of a stock solution (10 mg/mL) of Alexa Fluor 555 NHS ester
was added to the mixture. The reaction lasted for 1 h in the dark
at room temperature. Unconjugated dye molecules were then removed
by Zeba Dye and Biotin Removal Filter Plates (Thermo Fisher Scientific,
Waltham, MA, USA). The reference material, NIST human serum 909c (Millipore
Sigma, Darmstadt, Germany), was fluorescently labeled with Alexa Fluor
647 NHS ester (Thermo Fisher Scientific, Waltham, MA, USA) similarly.
The amounts of reagents were scaled linearly to the starting protein
amount (4 mg). Finally, each Alexa Fluor 555-labeled sample (10 μg
of total protein) was mixed with a proper volume of Alexa Fluor 647-labeled
reference material containing the same amount of protein, and the
final volume was adjusted to 50 μL with PBS.
concentrations of serum samples were measured with a DC protein assay
kit (Bio-Rad Laboratories, Hercules, CA, USA). Each volunteer serum
sample was fluorescently labeled with Alexa Fluor 555 NHS ester (Thermo
Fisher Scientific, Waltham, MA, USA). First, 10 μg of total
protein was diluted in PBS to 27 μL. The pH of the solution
was adjusted with 3 μL of 1 M sodium bicarbonate. Then 0.21
μL of a stock solution (10 mg/mL) of Alexa Fluor 555 NHS ester
was added to the mixture. The reaction lasted for 1 h in the dark
at room temperature. Unconjugated dye molecules were then removed
by Zeba Dye and Biotin Removal Filter Plates (Thermo Fisher Scientific,
Waltham, MA, USA). The reference material, NIST human serum 909c (Millipore
Sigma, Darmstadt, Germany), was fluorescently labeled with Alexa Fluor
647 NHS ester (Thermo Fisher Scientific, Waltham, MA, USA) similarly.
The amounts of reagents were scaled linearly to the starting protein
amount (4 mg). Finally, each Alexa Fluor 555-labeled sample (10 μg
of total protein) was mixed with a proper volume of Alexa Fluor 647-labeled
reference material containing the same amount of protein, and the
final volume was adjusted to 50 μL with PBS.
4
Fluorescent Protein Labeling for Biomolecular Interactions
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S-CMHumFt was labeled with Fluorescein-isothiocyanide (FITC) (Sigma Aldrich) according to the manufacturer’s standard protocol. Briefly, 2 mg/mL of purified protein were equilibrated in carbonate buffer pH 9.0. 50 µL of FITC 1 mg/mL in DMSO were added to 1 mL of S-CMHumFt solution and the reaction mix was stirred for 2 hours at room temperature. The non-reacted dye was removed by gel filtration chromatography and the labeled protein was dialyzed versus 20 mM HEPES buffer pH 7.4 containing 50 mM MgCl2.
Equine hearth cytochrome C (Cyt C) (Sigma Aldrich) was labeled with Alexa Fluor 555 NHS Ester (Succinimidyl Ester) (Thermo Fisher Scientific) according to the manufacturer’s standard protocol. Briefly, 10 mg of the protein were dissolved in 1 mL 0.1 M sodium bicarbonate buffer pH 8.4. 1 mg of Alexa Fluor 555 NHS Ester dissolved in 0.1 mL of DMSO was added dropwise to cytochrome C solution. The reaction was incubated in the dark for 1 hour at room temperature with continuous stirring. The non-reacted dye was removed by gel filtration chromatography and the labeled protein was dialyzed versus 20 mM HEPES buffer pH 7.4 containing 100 mM MgCl2.
Protein labeling was checked by High Performance Size Exclusion Chromatography (HP-SEC).
Equine hearth cytochrome C (Cyt C) (Sigma Aldrich) was labeled with Alexa Fluor 555 NHS Ester (Succinimidyl Ester) (Thermo Fisher Scientific) according to the manufacturer’s standard protocol. Briefly, 10 mg of the protein were dissolved in 1 mL 0.1 M sodium bicarbonate buffer pH 8.4. 1 mg of Alexa Fluor 555 NHS Ester dissolved in 0.1 mL of DMSO was added dropwise to cytochrome C solution. The reaction was incubated in the dark for 1 hour at room temperature with continuous stirring. The non-reacted dye was removed by gel filtration chromatography and the labeled protein was dialyzed versus 20 mM HEPES buffer pH 7.4 containing 100 mM MgCl2.
Protein labeling was checked by High Performance Size Exclusion Chromatography (HP-SEC).
5
Fluorescent Phage Labeling Protocol
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Phages were diluted in 0.1 M of sodium bicarbonate at a concentration of 4 × 1012 particles/ml. Phage solutions were incubated with Alexa Fluor 555 NHS Ester (Succinimidyl Ester, Thermo Fisher Scientific) at a concentration of 100 μM for 2 h at room temperature protected from light. Fluorescent phages were extensively dialyzed using dialysis buffer and dialysis tubing as described above. Phage fluorescence was confirmed by immunofluorescence microscopy before experimental use.
6
Glycan Array Analysis of Protein Binding
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The glycan array preparation and analysis was based on the method previously published18 (link). Briefly, BOry was labeled with Alexa Fluor 555 NHS ester (Thermo Fischer Scientific) following manufacturer instructions and YOry binding was determined after incubation with rabbit-anti-His antibody (GenScript, Piscataway, NJ, USA) and Alexa Fluor-555 goat anti-rabbit IgG (Thermo Fischer Scientific). rOrysata (0.5 nM) was incubated in the microarray chip printed with 144 different glycans (Fig. S1 ). After washing, the fluorescence signal was analyzed on an Agilent G265BA microarray scanner system (Agilent Technologies, Santa Clara, CA, USA). Quantification of the signals was performed with ProScanArray Express (Perkin Elmer, Waltham, MA, USA) and Microsoft Excel software. The average of mean RFU values after background subtraction and standard deviation for four replicate spots was calculated. Average RFU values for each data set were normalized to the highest RFU value and represented as histograms employing Prism 6 software v6.07 (GraphPad, San Diego, CA, USA) (https://www.graphpad.com/ ).
7
Immunofluorescence Staining of Cytoskeletal Proteins
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Antibodies were α-tubulin (ab18251, Abcam), detyrosinated α-tubulin (ab43389, Abcam), β-tubulin (T7816, Sigma), β-actin (A5441, Sigma), NM2A (909801, BioLegend), NM2B (909901, BioLegend), Alexa Fluor 568 goat anti-mouse (A-11004, ThermoFisher Scientific), Alexa Fluor 568 goat anti-rabbit (A-11011, ThermoFisher Scientific), Alexa Fluor 488 goat anti-mouse (A32723, ThermoFisher Scientific), Alexa Fluor 488 goat anti-rabbit (A-11034, ThermoFisher Scientific). Phalloidin (ab176753, Abcam), DAPI (D3571, Invitrogen), and Alexa Fluor 555 NHS ester (A37571, ThermoFisher Scientific).
8
Fluorophore Conjugation of Antibodies
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In each conjugation reaction, 5 μg of antibody in PBS (BSA-free) is mixed with 1 μl 0.33 mM CF750 Dye SE/TFP esters (Biotium 92142), Alexa Fluor 647 NHS Ester (Thermo Fisher Scientific A20006), Alexa Fluor 555 NHS Ester (Thermo Fisher Scientific A20009), or Alexa Fluor 488 NHS Ester (Thermo Fisher Scientific A20000) in DMSO, and incubated at room temperature for 16 hours. Fluorophore-conjugated antibodies were then purified with 30 kDa molecular weight cutoff Amicon Ultra-0.5 Centrifugal Filter Unit (Millipore Sigma UFC5030BK) by adding 500 μl PBS and centrifugation at 13,000 g for 5 minutes twice. Purified fluorophore-conjugated antibodies were collected by centrifugation at 2,000 g for 2 minutes and adjusted to 0.1 mg/ml in PBS for short-term storage at 4°C. Antibodies are listed in Supplementary Table 5 .
9
Fluorescent Labeling of Recombinant Proteins
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Recombinant StrepII-tagged hItln1 and MBL were directly conjugated with fluorophores using NHS-ester conjugation chemistry. HItln1 was labeled with Alexa Fluor 647 NHS Ester (Succinimidyl Ester) (Thermo Fisher Scientific), and MBL was labeled with Alexa Fluor 555 NHS Ester (Succinimidyl Ester) (Thermo Fisher Scientific) following the manufacturer’s protocol as follows: Proteins were labeled in storage buffer [Hepes-EDTA (pH 7.4) as described above] by the addition of dye dissolved in dimethyl sulfoxide at a ratio of 1:4 (w:w, dye:protein) and incubation for 1 hour at room temperature with mixing. Unreacted dye was removed by desalting using a Zeba 2 ml 7 K MWCO Spin Desalting Column (Thermo Fisher Scientific) following the manufacturer’s protocol. Molar ratio of fluorophore: Protein was determined from ultraviolet-visible absorbance spectrum using the extinction coefficients: ε (Alexa Fluor 555) = 155,000 cm−1 M−1 and ε (Alexa Fluor 647) = 270,000 cm−1 M−1.
10
Lectin-mediated Cell Glycan Analysis
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Chemicals were purchased from Sigma–Aldrich or Acros Organics, and specific amines from Key Organics, Crea‐Chim, Vitas M Labs, Life Chemicals, Alinda Chemicals, Chem Bridge, or Enamine BB (suppliers indicated in the Supporting Information, Characterization of the ligands) and were used without further purification. All reaction solvents were dried over activated 4 or 3 Å molecular sieves. TLC was carried out using 60 F254 TLC plates and visualized by UV irradiation (254 nm) or by staining with cerium molibdate, potassium permanganate, or ninhydrin solution. Canavalia ensiformis lectin (ConA) and Aleuria aurantia lectin (AAL) were purchased from VectorLabs and labeled with Alexa Fluor® 555 NHS Ester (Succinimidyl Ester) (Thermo Fisher Scientific). Human CLRs were prepared and labeled as described below.
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