mFluor™ UV610 SE

<strong>Top)</strong> Spectral pattern was generated using a 4-laser spectral cytometer. Spatially offset lasers (355 nm, 405 nm, 488 nm, and 640 nm) were used to generate four distinct emission profiles, then, when combined, yielded the overall spectral signature. <strong>Bottom)</strong> Flow cytometry analysis of whole blood cells stained with CD4-mFluor™ UV610 conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the mFluor™ UV610 specific UV10-A channel.

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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Physical properties

Molecular weight	1692.11
Solvent	DMSO

Spectral properties

Absorbance (nm)	589
Correction Factor (260 nm)	0.949
Correction Factor (280 nm)	0.904
Extinction coefficient (cm ^-1 M ^-1)	90000¹
Excitation (nm)	590
Emission (nm)	609
Quantum yield	0.25

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12171501

Overview SDS Protocol

Molecular weight

1692.11

Absorbance (nm)

589

Correction Factor (260 nm)

0.949

Correction Factor (280 nm)

0.904

Extinction coefficient (cm ^-1 M ^-1)

90000¹

Excitation (nm)

590

Emission (nm)

609

Quantum yield

0.25

mFluor™ dyes are developed for multicolor flow cytometry-focused applications. These dyes have large Stokes Shifts, and can be well excited by the laser lines of flow cytometers (e.g., 350 nm, 405 nm, 488 nm and 633 nm). mFluor™ UV dyes are optimized to be excited with a UV laser at 350 nm. AAT Bioquest offers the largest collection of fluorescent dyes that are excited by UV laser at 350 nm. mFluor™ UV 610 dyes have fluorescence excitation and emission maxima of ~350 nm and ~610 nm respectively. These spectral characteristics make them a unique color for flow cytometry application. mFluor™ UV 610 SE is reasonably stable and shows good reactivity and selectivity with protein amino groups. mFluor™ UV 610 SE provides a convenient tool to label monoclonal, polyclonal antibodies or other proteins (>10 kDa) for flow cytometric applications with the UV laser excitation.

Example protocol

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Protein stock solution (Solution A)

Mix 100 µL of a reaction buffer (e.g., 1 M sodium carbonate solution or 1 M phosphate buffer with pH ~9.0) with 900 µL of the target protein solution (e.g. antibody, protein concentration >2 mg/mL if possible) to give 1 mL protein labeling stock solution.
Note     The pH of the protein solution (Solution A) should be 8.5 ± 0.5. If the pH of the protein solution is lower than 8.0, adjust the pH to the range of 8.0-9.0 using 1 M sodium bicarbonate solution or 1 M pH 9.0 phosphate buffer.
Note     The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4. If the protein is dissolved in Tris or glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation.
Note     Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well. The presence of sodium azide or thimerosal might also interfere with the conjugation reaction. Sodium azide or thimerosal can be removed by dialysis or spin column for optimal labeling results.
Note     The conjugation efficiency is significantly reduced if the protein concentration is less than 2 mg/mL. For optimal labeling efficiency the final protein concentration range of 2-10 mg/mL is recommended.

2. mFluor™ UV610 SE stock solution (Solution B)

Add anhydrous DMSO into the vial of mFluor™ UV610 SE to make a 10 mM stock solution. Mix well by pipetting or vortex.
Note Prepare the dye stock solution (Solution B) before starting the conjugation. Use promptly. Extended storage of the dye stock solution may reduce the dye activity. Solution B can be stored in freezer for two weeks when kept from light and moisture. Avoid freeze-thaw cycles.

SAMPLE EXPERIMENTAL PROTOCOL

This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with mFluor™ UV610 SE. You might need further optimization for your particular proteins.
Note Each protein requires distinct dye/protein ratio, which also depends on the properties of dyes. Over labeling of a protein could detrimentally affects its binding affinity while the protein conjugates of low dye/protein ratio gives reduced sensitivity.

Run conjugation reaction

Use 10:1 molar ratio of Solution B (dye)/Solution A (protein) as the starting point: Add 5 µL of the dye stock solution (Solution B, assuming the dye stock solution is 10 mM) into the vial of the protein solution (95 µL of Solution A) with effective shaking. The concentration of the protein is ~0.05 mM assuming the protein concentration is 10 mg/mL and the molecular weight of the protein is ~200KD.
Note We recommend to use 10:1 molar ratio of Solution B (dye)/Solution A (protein). If it is too less or too high, determine the optimal dye/protein ratio at 5:1, 15:1 and 20:1 respectively.
Continue to rotate or shake the reaction mixture at room temperature for 30-60 minutes.

Purify the conjugation

The following protocol is an example of dye-protein conjugate purification by using a Sephadex G-25 column.

Prepare Sephadex G-25 column according to the manufacture instruction.
Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Combine the fractions that contain the desired dye-protein conjugate.
Note For immediate use, the dye-protein conjugate need be diluted with staining buffer, and aliquoted for multiple uses.
Note For longer term storage, dye-protein conjugate solution need be concentrated or freeze dried.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of mFluor™ UV610 SE to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

	0.1 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	59.098 µL	295.489 µL	590.978 µL	2.955 mL	5.91 mL
5 mM	11.82 µL	59.098 µL	118.196 µL	590.978 µL	1.182 mL
10 mM	5.91 µL	29.549 µL	59.098 µL	295.489 µL	590.978 µL

Molarity calculator

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Spectrum

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Spectral properties

Absorbance (nm)	589
Correction Factor (260 nm)	0.949
Correction Factor (280 nm)	0.904
Extinction coefficient (cm ^-1 M ^-1)	90000¹
Excitation (nm)	590
Emission (nm)	609
Quantum yield	0.25

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
mFluor™ UV375 SE	351	387	30000¹	0.94¹	0.099	0.138
mFluor™ UV460 SE	358	456	15000¹	0.86¹	0.35	0.134
mFluor™ UV420 SE	353	421	75000¹	-	-	-
mFluor™ UV455 SE	357	461	20000¹	0.42¹	0.651	0.406
mFluor™ UV520 SE	503	524	80000¹	-	0.495	0.518
mFluor™ UV540 SE	542	560	90000¹	0.35¹	0.634	0.463

Images

Figure 1. Top) Spectral pattern was generated using a 4-laser spectral cytometer. Spatially offset lasers (355 nm, 405 nm, 488 nm, and 640 nm) were used to generate four distinct emission profiles, then, when combined, yielded the overall spectral signature. Bottom) Flow cytometry analysis of whole blood cells stained with CD4-mFluor™ UV610 conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the mFluor™ UV610 specific UV10-A channel.

Fluorescent dye NHS esters (or succinimidyl esters) are the most popular tool for conjugating dyes to a peptide, protein, antibody, amino-modified oligonucleotide, or nucleic acid. NHS esters react readily with the primary amines (R-NH<sub>2</sub>) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting dye conjugates are quite stable.

Figure 2. Fluorescent dye NHS esters (or succinimidyl esters) are the most popular tool for conjugating dyes to a peptide, protein, antibody, amino-modified oligonucleotide, or nucleic acid. NHS esters react readily with the primary amines (R-NH₂) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting dye conjugates are quite stable.

References

View all 41 references: Citation Explorer

Deep ultraviolet lasers for flow cytometry.
Authors: Telford, William and Georges, Thierry and Miller, Clint and Voluer, Pascal
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2019): 227-233

Ultraviolet 320 nm laser excitation for flow cytometry.
Authors: Telford, William and Stickland, Lynn and Koschorreck, Marco
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2017): 314-325

Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish.
Authors: Pruitt, Margaret M and Marin, Wilfredo and Waarts, Michael R and de Jong, Jill L O
Journal: Journal of visualized experiments : JoVE (2017)

Methylene blue inhibits the asexual development of vivax malaria parasites from a region of increasing chloroquine resistance.
Authors: Suwanarusk, Rossarin and Russell, Bruce and Ong, Alice and Sriprawat, Kanlaya and Chu, Cindy S and PyaePhyo, Aung and Malleret, Benoit and Nosten, François and Renia, Laurent
Journal: The Journal of antimicrobial chemotherapy (2015): 124-9

Near-ultraviolet laser diodes for brilliant ultraviolet fluorophore excitation.
Authors: Telford, William G
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2015): 1127-37

Isolation and time lapse microscopy of highly pure hepatic stellate cells.
Authors: Bartneck, Matthias and Warzecha, Klaudia Theresa and Tag, Carmen Gabriele and Sauer-Lehnen, Sibille and Heymann, Felix and Trautwein, Christian and Weiskirchen, Ralf and Tacke, Frank
Journal: Analytical cellular pathology (Amsterdam) (2015): 417023

Rapid preparation of rodent testicular cell suspensions and spermatogenic stages purification by flow cytometry using a novel blue-laser-excitable vital dye.
Authors: Rodríguez-Casuriaga, Rosana and Santiñaque, Federico F and Folle, Gustavo A and Souza, Elisa and López-Carro, Beatriz and Geisinger, Adriana
Journal: MethodsX (2014): 239-43

Live cell cycle analysis of Drosophila tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle violet DNA stain.
Authors: Flegel, Kerry and Sun, Dan and Grushko, Olga and Ma, Yiqin and Buttitta, Laura
Journal: Journal of visualized experiments : JoVE (2013): e50239

DyeCycle Violet used for side population detection is a substrate of P-glycoprotein.
Authors: Boesch, Maximilian and Reimer, Daniel and Rumpold, Holger and Zeimet, Alain G and Sopper, Sieghart and Wolf, Dominik
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2012): 517-22

Effects of menadione, hydrogen peroxide, and quercetin on apoptosis and delayed luminescence of human leukemia Jurkat T-cells.
Authors: Baran, Irina and Ganea, Constanta and Scordino, Agata and Musumeci, Francesco and Barresi, Vincenza and Tudisco, Salvatore and Privitera, Simona and Grasso, Rosaria and Condorelli, Daniele F and Ursu, Ioan and Baran, Virgil and Katona, Eva and Mocanu, Maria-Magdalena and Gulino, Marisa and Ungureanu, Raluca and Surcel, Mihaela and Ursaciuc, Cornel
Journal: Cell biochemistry and biophysics (2010): 169-79