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iFluor® 860 succinimidyl ester

In vivo fluorescence imaging uses a sensitive camera to detect the fluorescence emission from fluorophores in whole-body living small animals. To overcome the photon attenuation in living tissue, fluorophores with long emission at the infrared (IR) region are generally preferred. Recent advances in imaging strategies and reporter techniques for in vivo fluorescence imaging include novel approaches to improve the specificity and affinity of the probes and to modulate and amplify the signal at target sites for enhanced sensitivity. Further emerging developments aim to achieve high-resolution, multimodality, and lifetime-based in vivo fluorescence imaging. Our iFluor® 860 is designed to label proteins and other biomolecules with infrared fluorescence. Conjugates prepared with iFluor® 860 have excitation and emission in the IR range. iFluor® 860 dye emission is well separated from commonly used far-red fluorophores such as Cy5, Cy7, or allophycocyanin (APC), facilitating multicolor analysis. This fluorophore is also useful for small animal in vivo imaging applications or other imaging applications requiring IR detection. iFluor® 860 succinimidyl ester is amine-reactive and can be readily used to conjugate amine-containing biomolecules, particularly antibodies.

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. iFluor™ 860 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of iFluor™ 860 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 iFluor™ 860 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
  1. 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.
  2. 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.
  1. Prepare Sephadex G-25 column according to the manufacture instruction.
  2. Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
  3. Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
  4. 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 iFluor® 860 succinimidyl ester to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM63.617 µL318.086 µL636.173 µL3.181 mL6.362 mL
5 mM12.723 µL63.617 µL127.235 µL636.173 µL1.272 mL
10 mM6.362 µL31.809 µL63.617 µL318.086 µL636.173 µL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® 350 succinimidyl ester3454502000010.9510.830.23
iFluor® 405 succinimidyl ester4034273700010.9110.480.77
iFluor® 488 succinimidyl ester4915167500010.910.210.11
iFluor® 514 succinimidyl ester5115277500010.8310.2650.116
iFluor® 532 succinimidyl ester5375609000010.6810.260.16
iFluor® 555 succinimidyl ester55757010000010.6410.230.14
iFluor® 594 succinimidyl ester58760320000010.5310.050.04
iFluor® 633 succinimidyl ester64065425000010.2910.0620.044
iFluor® 647 succinimidyl ester65667025000010.2510.030.03
iFluor® 660 succinimidyl ester66367825000010.2610.070.08
iFluor® 680 succinimidyl ester68470122000010.2310.0970.094
iFluor® 700 succinimidyl ester69071322000010.2310.090.04
iFluor® 750 succinimidyl ester75777927500010.1210.0440.039
iFluor® 610 succinimidyl ester61062811000010.8510.320.49
iFluor® 710 succinimidyl ester71673915000010.6010.120.07
iFluor® 790 succinimidyl ester78781225000010.1310.10.09
iFluor® 800 succinimidyl ester80182025000010.1110.030.08
iFluor® 810 succinimidyl ester81182225000010.0510.090.15
iFluor® 820 succinimidyl ester82285025000010.110.16
iFluor® 546 succinimidyl ester54155710000010.6710.250.15
iFluor® 568 succinimidyl ester56858710000010.5710.340.15
iFluor® 430 succinimidyl ester4334984000010.7810.680.3
iFluor® 450 succinimidyl ester4515024000010.8210.450.27
iFluor® 840 succinimidyl ester8368792000001-0.20.09
iFluor® 560 succinimidyl ester56057112000010.5710.04820.069
iFluor® 670 succinimidyl ester67168220000010.5510.030.033
iFluor® 460 succinimidyl ester468493800001~0.810.980.46
iFluor® 440 succinimidyl ester4344804000010.6710.3520.229
iFluor® 665 succinimidyl ester667692110,00010.2210.120.09
iFluor® 690 succinimidyl ester68570422000010.3010.090.06
iFluor® 720 succinimidyl ester71674024000010.1410.150.13
iFluor® 740 succinimidyl ester74076422500010.2010.160.16
iFluor® 597 succinimidyl ester59861810000010.710.3350.514
iFluor® 770 succinimidyl ester77779725000010.160.090.08
iFluor® 780 succinimidyl ester78480825000010.1610.130.12
iFluor® 570 succinimidyl ester55757012000010.581--
iFluor® 830 succinimidyl ester830867----
iFluor® 675 succinimidyl ester683700---0.066
iFluor® 620 succinimidyl ester621636---0.04
iFluor® 605 succinimidyl ester603623----
iFluor® 625 succinimidyl ester624640----
iFluor® 510 succinimidyl ester511530----
iFluor® 540 succinimidyl ester540557---0.105
iFluor® 445 succinimidyl ester446558----
iFluor® 500 succinimidyl ester501520----
Show More (36)

Citations

View all 4 citations: Citation Explorer
Identification of distinct functional thymic programming of fetal and pediatric human $\gamma$$\delta$ thymocytes via single-cell analysis
Authors: Sanchez Sanchez, Guillem and Papadopoulou, Maria and Azouz, Abdulkader and Tafesse, Yohannes and Mishra, Archita and Chan, Jerry KY and Fan, Yiping and Verdebout, Isoline and Porco, Silvana and Libert, Fr{\'e}d{\'e}rick and others,
Journal: Nature communications (2022): 1--19
Gas plasma irradiation of breast cancers promotes immunogenicity, tumor reduction, and an abscopal effect in vivo
Authors: Mahdikia, Hamed and Saadati, Fariba and Freund, Eric and Gaipl, Udo S and Majidzadeh-a, Keivan and Shokri, Babak and Bekeschus, Sander
Journal: OncoImmunology (2021): 1859731
Nanovesicle delivery to the liver via retinol binding protein and platelet-derived growth factor receptors: how targeting ligands affect biodistribution
Authors: Hsu, Ching-Yun and Chen, Chun-Han and Aljuffali, Ibrahim A and Dai, You-Shan and Fang, Jia-You
Journal: Nanomedicine (2017)

References

View all 18 references: Citation Explorer
In Vivo Fluorescence Microscopic Imaging for Dynamic Quantitative Assessment of Intestinal Mucosa Permeability in Mice
Authors: Szabo A, Vollmar B, Boros M, Menger MD.
Journal: J Surg Res. (2007)
A target cell-specific activatable fluorescence probe for in vivo molecular imaging of cancer based on a self-quenched avidin-rhodamine conjugate
Authors: Hama Y, Urano Y, Koyama Y, Kamiya M, Bernardo M, Paik RS, Shin IS, Paik CH, Choyke PL, Kobayashi H.
Journal: Cancer Res (2007): 2791
Fluorescence imaging in vivo: recent advances
Authors: Rao J, Dragulescu-Andrasi A, Yao H.
Journal: Curr Opin Biotechnol (2007): 17
Ex vivo fluorescence imaging of normal and malignant urothelial cells to enhance early diagnosis
Authors: Steenkeste K, Lecart S, Deniset A, Pernot P, Eschwege P, Ferlicot S, Leveque-Fort S, Bri and et R, Fontaine-Aupart MP.
Journal: Photochem Photobiol (2007): 1157
In vivo monitoring the fate of Cy5.5-Tat labeled T lymphocytes by quantitative near-infrared fluorescence imaging during acute brain inflammation in a rat model of experimental autoimmune encephalomyelitis
Authors: Berger C, Gremlich HU, Schmidt P, Cannet C, Kneuer R, Hiest and P, Rausch M, Rudin M.
Journal: J Immunol Methods (2007): 65
Page updated on October 8, 2024

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Physical properties

Molecular weight

1571.90

Solvent

DMSO

Spectral properties

Correction Factor (260 nm)

0.1

Correction Factor (280 nm)

0.14

Extinction coefficient (cm -1 M -1)

2500001

Excitation (nm)

853

Emission (nm)

878

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12171501
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 (e.g., iFluor 860-antibodies) are quite stable.
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 (e.g., iFluor 860-antibodies) are quite stable.
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 (e.g., iFluor 860-antibodies) are quite stable.
Spectral signature of iFluor® 860 dye. Data acquired on a 4-laser Cytek Aurora and normal human peripheral blood cells stained with clone SK3 (CD4) conjugated to iFluor® 860 dye (Cat. No. 100420R0) were used for analysis.