Fluorescent Dye

Henna dyes are widely used for labeling cells, organelles, toners, viruses and lipoproteins. Long-chain henna cyanines include DiO (DiOC18 (3)), DiI (DiIC18 (3)), DiD (DiIC18 (5) )) and DiR, as well as the dialkylphoenix dye DiA (4-Di-16-ASP) Used to mark membranes and other focal structures. DiIC16 (3) has a shorter alkyl substituent (C16) than DiI (C18). They have extremely high extinction factors, environment-dependent fluorophores, and short excited-state depletion in pyramid environments. They are oil-forming and weakly fluorescent in water, but are highly fluorescent and fairly photostable when incorporated into membranes or bound to lipophilic biomolecules. These optical properties make them ideal for staining cell plasma membranes. Once cells are identified, these dyes diffuse laterally within the plasma membrane, resulting in staining of the entire cell [ 1 DiO, DiI, DiD, and DiR exhibit distinct green, orange, red, and red fluorescence, respectively, thereby facilitating multicolor imaging of live cells and Flow cytometry analysis. DiO and DiI can be used with standard FITC and TRITC filters respectively. Among them, DiI and its analogs are the most commonly used because they usually exhibit very low cytotoxicity. In addition, DiI is widely used to measure lipoproteins such as LDL and HDL. The lipophilic radical dye DiA is also commonly used to determine metatracing [2].

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Experiment protocol:
1. Preparation of Di Staining Solution
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a. Preparation of stock solution:
• Prepare 1-5 mM stock solution using dimethylformamide (DMF), dimethyl sulfoxide (DMSO) or ethanol
• DMF is preferred over ethanol as solvent
• Aliquot unused solution and store at -20°C
• Avoid repeated freeze-thaw cycles
• Storage period: 6 months
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b. Preparation of working solution:
• Dilute with serum-free medium, HBSS or PBS buffer to 1-5 μM
• Aqueous working solution should be freshly prepared and used within 24 hours
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2. Staining Procedure for Suspension Cells
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a. Cell pretreatment:
• Centrifuge at 1000g, 4°C for 3-5 minutes
• Wash twice with PBS, 5 minutes each
• Adjust cell density to 1×10⁶/mL
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b. Staining process:
• Add 1 mL Di working solution
• Incubate at room temperature for 5-30 minutes
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c. Post-treatment:
• Centrifuge at 400g, 4°C for 3-4 minutes
• Wash twice with PBS, 5 minutes each
• Resuspend in serum-free medium or PBS
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d. Detection:
• Analyze by fluorescence microscopy or flow cytometry
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3. Staining Procedure for Adherent Cells
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a. Cell preparation:
• Culture adherent cells on sterile coverslips
• Remove medium and aspirate excess liquid
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b. Staining process:
• Add 100 μL working solution
• Gently shake to ensure complete coverage of cells
• Incubate at room temperature for 5-30 minutes
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c. Post-treatment:
• Wash twice with fresh medium, 5 minutes each
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d. Detection:
• Analyze by fluorescence microscopy or flow cytometry
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Notes:
1. All operations should be performed protected from light
2. Unstained cell controls are recommended
3. Staining time can be optimized according to experimental conditions

DiI-labeled motor neurons can survive up to 4 weeks in culture and up to 1 year in vivo [1].

- Neuronal labeling in culture: Motoneurons isolated from adult frog spinal cords were cultured in vitro. DiI (DiIC18(3)) was added to the culture medium (concentration not specified). The dye integrated into the cell membranes, allowing visualization of neuronal somata, dendrites, and axons under fluorescence microscopy. Labeling remained stable for the entire culture period (up to several weeks), enabling long-term observation of neurite sprouting and morphological changes [2]
- Multicolor nervous tissue labeling: Fixed nervous system tissues (e.g., brain slices) were labeled using the "DiOlistic" method. DiI was coated onto gold particles, which were propelled into the tissue using a gene gun. The dye diffused along neuronal membranes, specifically labeling individual neurons. Combinations of DiI with other lipophilic dyes enabled multicolor visualization of adjacent neurons, facilitating analysis of neuronal networks [1]
- Stem cell tracking in 3D scaffolds: Mesenchymal stem cells (MSCs) were incubated with DiI (10 μM) for 30 minutes at 37°C, then seeded into 3D collagen scaffolds. Fluorescence microscopy showed that DiI efficiently labeled MSCs, with stable fluorescence for up to 21 days, allowing tracking of cell distribution and survival within the scaffold [3]
- Nanoparticle internalization assay: Cells were incubated with DiI-labeled nanoparticles (concentration adjusted based on nanoparticle type) for 1-24 hours. Confocal microscopy revealed the intracellular localization of nanoparticles, with DiI fluorescence (excitation 549 nm, emission 565 nm) used to quantify internalization efficiency and compare uptake mechanisms across different nanoparticle corona compositions [4]

- Myocardial infarction model: Rats with induced myocardial infarction were injected with DiI-labeled MSCs (labeled as in cell assay) via tail vein. Hearts were harvested at 1, 2, and 4 weeks post-injection. Immunofluorescence staining showed DiI-positive MSCs in the infarcted area, with persistence for up to 4 weeks, indicating cell retention and potential paracrine effects [3]

[1]. Multicolor "DiOlistic" labeling of the nervous system using lipophilic dye combinations. Neuron. 2000 Aug;27(2):219-25.

[2]. Long-term survival and sprouting in culture by motoneurons isolated from the spinal cord of adult frogs. J Comp Neurol. 1990 Dec 22;302(4):729-38.

[3]. Combination of mesenchymal stem cells and three-dimensional collagen scaffold preserves ventricular remodeling in rat myocardial infarction model. World J Stem Cells. 2022 Aug 26;14(8):633-657

[4]. Corona Composition Can Affect the Mechanisms Cells Use to Internalize Nanoparticles. ACS Nano. 2019 Oct 22;13(10):11107-11121.

- DiI (DiIC18(3)) is a lipophilic carbocyanine dye that embeds into lipid bilayers, exhibiting red fluorescence (excitation ~549 nm, emission ~565 nm). Its primary application is as a cell membrane tracer due to its ability to diffuse laterally within membranes without transferring between cells [1][2][3][4]
- The dye is widely used for labeling neurons, stem cells, and nanoparticles in vitro and in vivo, as it is non-toxic at working concentrations (≤20 μM) and maintains stable fluorescence in both fixed and live samples [2][3][4]

C59H97CLN2O4

933.89

932.714

C, 75.88; H, 10.47; Cl, 3.80; N, 3.00; O, 6.85

41085-99-8

2762626

Solid powder

68ºC (dec.)(lit.)

19.061

0

5

36

66

1210

0

CCCCCCCCCCCCCCCCCCN1C2=CC=CC=C2C(C1=CC=CC3=[N+](C4=CC=CC=C4C3(C)C)CCCCCCCCCCCCCCCCCC)(C)C.[O-]Cl(=O)(=O)=O

JVXZRNYCRFIEGV-UHFFFAOYSA-M

InChI=1S/C59H97N2.ClHO4/c1-7-9-11-13-15-17-19-21-23-25-27-29-31-33-35-41-50-60-54-46-39-37-44-52(54)58(3,4)56(60)48-43-49-57-59(5,6)53-45-38-40-47-55(53)61(57)51-42-36-34-32-30-28-26-24-22-20-18-16-14-12-10-8-2;2-1(3,4)5/h37-40,43-49H,7-36,41-42,50-51H2,1-6H3;(H,2,3,4,5)/q+1;/p-1

2-[3-(3,3-dimethyl-1-octadecylindol-1-ium-2-yl)prop-2-enylidene]-3,3-dimethyl-1-octadecylindole;perchlorate

DiI stain; DiIC18(3); 41085-99-8; 1,1'-Di-n-octadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; (2Z)-2-[(E)-3-(3,3-Dimethyl-1-octadecylindol-1-ium-2-yl)prop-2-enylidene]-3,3-dimethyl-1-octadecylindole;perchlorate; 2-[3-(3,3-dimethyl-1-octadecylindol-1-ium-2-yl)prop-2-enylidene]-3,3-dimethyl-1-octadecylindole;perchlorate; DiI perchlorate; DII; DiIC18(3); MFCD00142354;DiI

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO : ~12.5 mg/mL (~13.39 mM)

Solubility in Formulation 1: 1.25 mg/mL (1.34 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 1.25 mg/mL (1.34 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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