Fluorescent Dye
The Vari Fluor dyes do not target specific proteins or receptors by themselves; instead, they serve as optical imaging agents. Upon excitation with NIR light, the fluorophore emits photons that can be detected by sensitive cameras (e.g., CCD or InGaAs detectors) or flow cytometers. The carboxylic acid functional group provides a chemical handle for conjugation to targeting vectors, such as antibodies, peptides, or small molecules.

In live cells, Vari Fluor 750 has no intrinsic effect on cell viability or proliferation. The dye is non‑toxic and photostable, making it suitable for long‑term tracking experiments. When conjugated to a targeting vector (e.g., an anti‑EGFR antibody), the labeled construct can selectively label EGFR‑expressing cancer cells with high signal‑to‑background ratio in flow cytometry and fluorescence microscopy.

When conjugated to a targeting antibody or peptide, Vari Fluor 750 enables in vivo near‑infrared fluorescence imaging of tumors in mice. The dye is administered intravenously (0.1‑10 nmol per mouse), and the animals are imaged at various time points (1‑96 hours) using a small‑animal NIR imaging system. Because the NIR window allows light to penetrate several centimeters through tissue, the labeled probe can visualize deep‑seated tumors with high sensitivity.

The carboxylic acid group on Vari Fluor 750 must be activated before conjugation. Typically, the dye is dissolved in dry DMSO or DMF (10 mg/mL), and an activation cocktail containing 1‑ethyl‑3‑(3‑dimethylaminopropyl)carbodiimide (EDC) and N‑hydroxysuccinimide (NHS) (molar ratio 1:1:2, dye:EDC:NHS) is added. The mixture is incubated for 15‑30 minutes at room temperature. The active NHS ester thus formed is then reacted with primary amines (lysine residues or N‑termini) of the target protein in PBS (pH 7.4) for 1‑2 hours at room temperature.

No standard cell‑based assay is performed for the free acid form because the carboxylic acid does not enter cells efficiently and lacks intrinsic bioactivity. Instead, after conjugation to a cell‑penetrating peptide or an antibody, the labeled product is tested by flow cytometry or confocal microscopy. Cells are incubated with the labeled conjugate (1‑10 ug/mL) for 30‑60 minutes at 4degC or 37degC, washed, and analyzed by flow cytometry (FL4 channel, 740‑780 nm emission).

For in vivo imaging, a xenograft mouse model is established by subcutaneous injection of 1‑5×10⁶ tumor cells into the flank of nude mice. When tumors reach 5‑10 mm in diameter, the Vari Fluor 750‑labeled targeting probe (0.1‑10 nmol) is injected intravenously via the tail vein. Mice are anesthetized with isoflurane and imaged using an IVIS Spectrum or similar NIR fluorescence imaging system at 1, 4, 8, 24, and 48 hours post‑injection. Regions of interest are drawn around the tumor, and fluorescence intensity is quantified.

As a small molecule (MW ~800‑1000 g/mol), Vari Fluor 750 carboxylic acid is highly lipophilic and albumin‑bound in plasma. It clears relatively slowly from the circulation (half‑life 4‑12 hours) and accumulates in the liver and spleen, where it is eventually metabolized and excreted via the bile and urine. The free acid form is not orally bioavailable and must be injected.

The Vari Fluor 750 chromophore itself is considered non‑toxic and non‑genotoxic at the low doses used for imaging (typically <10 ug per mouse). However, the activating agents (EDC, NHS) and organic solvents (DMSO, DMF) used for conjugation can be toxic, and careful removal of these reagents is necessary before in vivo administration. No specific LD50 has been determined.

[1]. A unique class of near-infrared functional fluorescent dyes with carboxylic-acid-modulated fluorescence ON/OFF switching: rational design, synthesis, optical properties, theoretical calculations, and applications for fluorescence imaging in living animals. J Am Chem Soc. 2012 Jan 18;134(2):1200-11.

Fluorescence imaging is one of the most powerful techniques for monitoring biomolecules in biological systems. Near-infrared (NIR) fluorescence sensors, with their absorption and emission spectra in the near-infrared region, are well-suited for bioimaging applications in live animals due to the advantages of low photodamage, deep tissue penetration, and minimal interference from background autofluorescence. This paper proposes a novel strategy for designing near-infrared functional dyes with a carboxylic acid-controlled fluorescence switching mechanism via spirocyclization. Based on this design strategy, we developed a series of Changsha (CS1-6) near-infrared fluorescent dyes, a unique class of novel near-infrared functional fluorescent dyes with excellent photophysical properties, including large absorption extinction coefficients, high fluorescence quantum yields, high brightness, good photostability, and sufficient chemical stability. Notably, the novel CS1-6 NIR dyes retain the rhodamine-like fluorescence switching mechanism while also exhibiting absorption and emission spectra in the near-infrared region, superior to traditional rhodamine-like dyes. Furthermore, we performed quantum chemical calculations using the 6-31G basis set and B3LYP exchange functional to elucidate the structure-optical properties of the novel CS1-6 NIR dyes. In addition, using CS2 as a platform, we further constructed a novel near-infrared fluorescence-on sensor 7, which can image HClO produced endogenously in living animals, demonstrating the value of our novel CS near-infrared functional fluorescent dye. We expect that this design strategy can be extended to the development of various near-infrared functional dyes with suitable fluorescence control mechanisms for many applications in biological research. [1]

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Vari Fluor 750 carboxylic acid is a research‑grade near‑infrared dye not approved for human use. It is part of a new generation of NIR dyes with higher photostability and brightness than older dyes such as Cy5.5 and Cy7. The carboxylic acid form is the preferred starting material for custom labeling of biomolecules. Its excitation/emission profile (747/770 nm) places it in the “optical window” for in vivo imaging, where tissue absorption and autofluorescence are minimal.

Light blue to blue Solid powder

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)