ICG-Sulfo-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

AT A GLANCE

Molecular Weight: 930.07
Solvent: DMSO
Extinction Coefficient: 230,000 cm-1M-1
Excitation/Emission: 780/800 nm
CF at 260/280 nm: 0.113/0.073
Important Extinction coefficient are at their maximum absorption wavelength. CF at 260 nm is the correction factor used for eliminating the dye contribution to the absorbance at 260 nm (for oligo and nucleic acid labeling). CF at 280 nm is the correction factor used for eliminating the dye contribution to the absorbance at 280 nm (for peptide and protein labeling). Fluorescence intensity is significantly increased upon coupled to proteins. This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with ICG. You might need further optimization for your particular proteins.

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. The pH of the protein solution 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. 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. 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. For optimal labeling efficiency, the final protein concentration range of 2 - 10 mg/mL is recommended, with the conjugation efficiency significantly reduced if less than 2 mg/mL.

2. Dye Stock Solution (Solution B)

Add anhydrous DMSO into the vial of ICG dyes to make a 10 - 20 mM stock solution. Mix well by pipetting or vortex. Prepare the dye stock solution before starting the conjugation. Use promptly. Extended storage of the dye stock solution may reduce dye activity.

SAMPLE EXPERIMENTAL PROTOCOL

Determine the optimal dye/protein ratio (optional):

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. The concentration of the DMSO in the protein solution should be <10%.
Run conjugation reaction.
Repeat Step 2 with the molar ratios of Solution B/Solution A at 5:1; 15:1 and 20:1 respectively.
Purify the desired conjugates using premade spin columns.
Calculate the dye/protein ratio (DOS) for the above 4 conjugates (if step 3 is done).
Run your functional tests of the above 4 conjugates to determine the best dye/protein ratio to scale up your labeling reaction.

Run conjugation reaction:

Add the appropriate amount of dye stock solution (Solution B) into the vial of the protein solution (Solution A) with effective shaking. The best molar ratio of Solution B/Solution is determined above. If skipped, we recommend using 10:1 molar ratio of Solution B (dye)/Solution A (protein).
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 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. For immediate use, the dye-protein conjugate need be diluted with staining buffer, and aliquoted for multiple uses. 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 ICG-Sulfo-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 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	107.519 µL	537.594 µL	1.075 mL	5.376 mL	10.752 mL
5 mM	21.504 µL	107.519 µL	215.038 µL	1.075 mL	2.15 mL
10 mM	10.752 µL	53.759 µL	107.519 µL	537.594 µL	1.075 mL

Molarity calculator

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

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

Spectrum

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Product family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Correction Factor (280 nm)	Correction Factor (260 nm)
ICG-PEG12-OSu	789	813	230000	0.076	-
ICG Xtra-OSu	790	814	230000	0.055	0.09

Citations

View all 17 citations: Citation Explorer

Targeted delivery of cathepsin-activatable near-infrared fluorescence probe for ultrahigh specific imaging of peritoneal metastasis
Authors: Lu, Jialiang and Guo, Yu and Hao, Huimin and Ma, Junjie and Lu, Yang and Sun, Yue and Shi, Zheng and Dong, Xiaowu and Zhang, Bo and Fang, Luo and others,
Journal: European Journal of Medicinal Chemistry (2023): 115909

pH-gated nanoparticles selectively regulate lysosomal function of tumour-associated macrophages for cancer immunotherapy
Authors: Tang, Mingmei and Chen, Binlong and Xia, Heming and Pan, Meijie and Zhao, Ruiyang and Zhou, Jiayi and Yin, Qingqing and Wan, Fangjie and Yan, Yue and Fu, Chuanxun and others,
Journal: Nature Communications (2023): 5888

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

Quantitative imaging of intracellular nanoparticle exposure enables prediction of nanotherapeutic efficacy
Authors: Yin, Qingqing and Pan, Anni and Chen, Binlong and Wang, Zenghui and Tang, Mingmei and Yan, Yue and Wang, Yaoqi and Xia, Heming and Chen, Wei and Du, Hongliang and others,
Journal: Nature communications (2021): 1--13

Detection of lymph node metastases by ultra-pH-sensitive polymeric nanoparticles
Authors: Bennett, Zachary T and Feng, Qiang and Bishop, Justin A and Huang, Gang and Sumer, Baran D and Gao, Jinming
Journal: Theranostics (2020): 3340

References

View all 193 references: Citation Explorer

Establishment of novel detection system for embryonic stem cell-derived hepatocyte-like cells based on nongenetic manipulation with indocyanine green
Authors: Yoshie S, Ito J, Shirasawa S, Yokoyama T, Fujimura Y, Takeda K, Mizuguchi M, Matsumoto K, Tomotsune D, Sasaki K.
Journal: Tissue Eng Part C Methods (2012): 12

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

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 November 21, 2024

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Physical properties

Molecular weight	930.07
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

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