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ICG-OSu

Indocyanine green (ICG) is a cyanine dye used in medical diagnostics. It is used for determining cardiac output, hepatic function, and liver blood flow, and for ophthalmic angiography. It has a peak spectral absorption close to 800 nm. These infrared frequencies penetrate retinal layers, allowing ICG angiography to image deeper patterns of circulation than fluorescein angiography. ICG binds tightly to plasma proteins and becomes confined to the vascular system. ICG has a half-life of 150 to 180 seconds and is removed from circulation exclusively by the liver to bile juice. A recent study indicated ICG targets atheromas within 20 min of injection and provides sufficient signal enhancement for in vivo detection of lipid-rich, inflamed, coronary-sized plaques in atherosclerotic rabbits. Ex vivo fluorescence reflectance imaging showed high plaque target-to-background ratios in atheroma-bearing rabbits injected with ICG compared to atheroma-bearing rabbits injected with saline. This amino-reactive ICG derivative is used to make ICG bioconjugates with antibodies and other biological molecules. It has moderate water solubility.

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

Protein stock solution (Solution A)
  1. 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.

ICG-OSu stock solution (Solution B)
  1. Add anhydrous DMSO into the vial of ICG-OSu 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 the 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 ICG-OSu. You might need further optimization for your particular proteins.

Note: Each protein requires a distinct dye/protein ratio, which also depends on the properties of dyes. Over-labeling of a protein could detrimentally affect its binding affinity while the protein conjugates of low dye/protein ratio give reduced sensitivity.

Run conjugation reaction
  1. Use a 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 using a 10:1 molar ratio of Solution B (dye)/Solution A (protein). If it is too low 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 the Sephadex G-25 column according to the manufacturer's instructions.

  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, the dye-protein conjugate solution needs to be concentrated or freeze-dried.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of ICG-OSu 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 mM120.769 µL603.843 µL1.208 mL6.038 mL12.077 mL
5 mM24.154 µL120.769 µL241.537 µL1.208 mL2.415 mL
10 mM12.077 µL60.384 µL120.769 µL603.843 µL1.208 mL

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)Correction Factor (280 nm)
ICG-ATT [3-ICG-acyl-1,3-thiazolidine-2-thione]7898132300000.076
ICG amine7898132300000.076
ICG acid7898132300000.076
ICG Maleimide7898132300000.076
ICG azide7898132300000.076
ICG alkyne7898132300000.076
ICG hydrazide7898132300000.076

Citations

View all 9 citations: Citation Explorer
Comparison of Near-Infrared Imaging Agents Targeting the PTPmu Tumor Biomarker
Authors: Johansen, Mette L and Vincent, Jason and Rose, Marissa and Sloan, Andrew E and Brady-Kalnay, Susann M
Journal: Molecular Imaging and Biology (2023): 1--14
Polymeric Nanoreactors with Chemically Tunable Redox Responsivity
Authors: Gong, Lidong and Wang, Changrong and Xu, Pengcheng and Gong, Jingjing and Zhu, Chuanda and Di, Shiming and Li, Yanglonghao and Mu, Yongxu and Han, Hongbin and Zhang, Qiang and others,
Journal: ACS Applied Materials \& Interfaces (2022): 40266--40275
Comparison of Five Near-Infrared Fluorescent Folate Conjugates in an Ovarian Cancer Model
Authors: Garc{\'\i}a de Jal{\'o}n, Elvira and Kleinmanns, Katrin and Fosse, Vibeke and Davidson, Ben and Bj{\o}rge, Line and Haug, Bengt Erik and McCormack, Emmet
Journal: Molecular Imaging and Biology (2021): 1--12
Self-assembly of four generations of RNA dendrimers for drug shielding with controllable layer-by-layer release
Authors: Li, Xin and Vieweger, Mario and Guo, Peixuan
Journal: Nanoscale (2020): 16514--16525
Nanoparticle Targeting of Neutrophil Improves Cancer Therapy
Authors: Shi, Xutong
Journal: (2018)

References

View all 193 references: Citation Explorer
Sentinel lymph node biopsy using intraoperative indocyanine green fluorescence imaging navigated with preoperative CT lymphography for superficial esophageal cancer
Authors: Yuasa Y, Seike J, Yoshida T, Takechi H, Yamai H, Yamamoto Y, Furukita Y, Goto M, Minato T, Nishino T, Inoue S, Fujiwara S, Tangoku A.
Journal: Ann Surg Oncol (2012): 486
Indocyanine green angiography-guided photodynamic therapy for treatment of chronic central serous chorioretinopathy: a pilot study
Authors: Yannuzzi LA, Slakter JS, Gross NE, Spaide RF, Costa DL, Huang SJ, Klancnik JM, Jr., Aizman A.
Journal: Retina (2012): 288
Using indocyanine green fluorescent lymphography and lymphatic-venous anastomosis for cancer-related lymphedema
Authors: Mihara M, Murai N, Hayashi Y, Hara H, Iida T, Narushima M, Todokoro T, Uchida G, Yamamoto T, Koshima I.
Journal: Ann Vasc Surg (2012): 278 e1
Indocyanine green fluorescence endoscopy for visual differentiation of pituitary tumor from surrounding structures
Authors: Litvack ZN, Zada G, Laws ER, Jr.
Journal: J Neurosurg. (2012)
Management of peripheral polypoidal choroidal vasculopathy with intravitreal bevacizumab and indocyanine green angiography-guided laser photocoagulation
Authors: Rishi P, Das A, Sarate P, Rishi E.
Journal: Indian J Ophthalmol (2012): 60
Page updated on December 6, 2024

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Physical properties

Molecular weight

828.03

Solvent

DMSO

Spectral properties

Correction Factor (280 nm)

0.076

Extinction coefficient (cm -1 M -1)

230000

Excitation (nm)

789

Emission (nm)

813

Storage, safety and handling

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

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12171501

CAS

1622335-40-3
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