DiO is a long-chain carbocyanine dye. Carbocyanine dyes are extensively used as lipophilic tracers for labeling cells, organelles, liposomes, viruses, and lipoproteins.

Physicochemical Properties

Molecular Formula	C53H85CLN2O6
Molecular Weight	881.70500
Exact Mass	880.61
CAS #	34215-57-1
PubChem CID	9962797
Appearance	Orange to red solid powder
LogP	16.78
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	36
Heavy Atom Count	62
Complexity	1020
Defined Atom Stereocenter Count	0
SMILES	CCCCCCCCCCCCCCCCCCN\1C2=CC=CC=C2O/C1=C/C=C/C3=[N+](C4=CC=CC=C4O3)CCCCCCCCCCCCCCCCCC.[O-]Cl(=O)(=O)=O
InChi Key	GFZPJHFJZGRWMQ-UHFFFAOYSA-M
InChi Code	InChI=1S/C53H85N2O2.ClHO4/c1-3-5-7-9-11-13-15-17-19-21-23-25-27-29-31-37-46-54-48-40-33-35-42-50(48)56-52(54)44-39-45-53-55(49-41-34-36-43-51(49)57-53)47-38-32-30-28-26-24-22-20-18-16-14-12-10-8-6-4-2;2-1(3,4)5/h33-36,39-45H,3-32,37-38,46-47H2,1-2H3;(H,2,3,4,5)/q+1;/p-1
Chemical Name	(2E)-3-octadecyl-2-[(E)-3-(3-octadecyl-1,3-benzoxazol-3-ium-2-yl)prop-2-enylidene]-1,3-benzoxazole;perchlorate
HS Tariff Code	2934.99.9001
Storage	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.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro	Labeling cells, organelles, toners, viruses, and lipoproteins is a common application for henna dyes. DiO (DiOC18 (3)), DiI (DiIC18 (3)), DiD (DiIC18 (5) )), and DiR are examples of long-chain henna cyanines. DiA (4-Di-16-ASP), a dialkylphoenix dye, is also used to mark membranes and other focal structures. Compared to DiI (C18), DiIC16 (3) has a shorter alkyl substituent (C16). They have fluorophores that are dependent on the environment, very high extinction factors, and short excited-state depletion in pyramid environments. When integrated into membranes or attached to lipophilic biomolecules, they become highly fluorescent and relatively photostable. In water, they are oil-forming and weakly fluorescent. They are perfect for staining cell plasma membranes because of their optical characteristics. After the cells are recognized, these dyes permeate laterally through the plasma membrane, staining the entire cell [1 DiO, DiI, DiD, and DiR show distinct green, orange, red, and red fluorescence, respectively, making it easier to image live cells in multiple colors and perform flow cytometry. Standard FITC and TRITC filters can be used with DiO and DiI, respectively. DiI and its analogs are the most widely used among them because of their typically extremely low cytotoxicity. DiI is also frequently used to measure lipoproteins, including HDL and LDL. For determination, the lipophilic radical dye DiA is also frequently utilized. First general protocol. Get the second staining solution ready. Prepare stock solutions in ethanol, dimethylformamide (DMF), or DMSO: Stock solutions should be represented at a concentration of 1–5 mM in ethanol, dimethylformamide (DMF), or DMSO. DMF contains a lot of ethanol and serves as a solvent for Di. It is recommended to aliquot stock solution as required. Aliquot any excess solution, then freeze it at or below -20°C. Do not freeze and thaw repeatedly. The solution has a six-month shelf life. To prepare a working solution, add the stock solution to the appropriate buffer. For instance, you can prepare a working solution of 1 to 5 μM using serum-free medium, HBSS, or PBS. We advise against keeping the storage solution in storage for longer than a day. Note: Working solution 2's ultimate concentration. Cells in suspension a. Centrifuge for three to five minutes at 1000g at 4°C. After washing twice for five minutes each time with PBS, discard the supernatant. The density of cells is 1×106/mL. b. After adding 1 mL of the Di working solution, view C. After centrifuging for three to four minutes at 400 g and 4°C, remove the supernatant. d. Use PBS to wash twice, for five minutes each time. e. Use PBS or serum-free cells to resuspend the cells, then use fluorescence microscopy for five to thirty minutes. 3. Cells that adhere a. On sterile coverslips, cultivate adherent cells. b. Clear the coverslip of the culture medium and use a tiny aspirator to extract a few cells. C. To fully cover the cells, add 100 μL of working solution and shake gently for a duration of 5-30 minutes. d. Apply culture medium twice, giving each wash five minutes. Use flow cytometry or fluorescence microscopy to observe. - 3,3'-Dioctadecyloxacarbocyanine perchlorate (DiOC18(3) perchlorate) is a lipophilic fluorescent probe that inserts into the lipid bilayer of model biological membranes via hydrophobic interactions. It exhibits characteristic photophysical properties: in polar solvents, it shows shorter fluorescence emission wavelength and lower quantum yield; in model biological membranes (e.g., liposomes), it displays red-shifted emission and higher fluorescence intensity, reflecting membrane environment polarity [3][4] - In cultured mouse soleus muscle cells, 3,3'-Dioctadecyloxacarbocyanine perchlorate labeled the plasma membrane. After contraction-induced injury, the probe redistributed from the membrane to the cytoplasm, which was detectable by fluorescence microscopy, indicating loss of membrane integrity [2] - In vitro primary neuronal cultures, 3,3'-Dioctadecyloxacarbocyanine perchlorate was used for multicolor "DiOlistic" labeling. It efficiently stained neuronal membranes and neurites, enabling clear visualization of neuronal morphology and branching when combined with other lipophilic dyes [1]
ln Vivo	- In mice, 3,3'-Dioctadecyloxacarbocyanine perchlorate was applied for DiOlistic labeling of the nervous system. Coated onto gold particles, the probe was delivered into brain and spinal cord tissues via gene gun. It specifically labeled neuronal membranes, allowing three-dimensional visualization of neural circuits and neurite projections [1] - In a mouse soleus muscle injury model, 3,3'-Dioctadecyloxacarbocyanine perchlorate was topically applied to the muscle after contraction-induced injury. In vivo fluorescence imaging showed probe redistribution in injured muscle fibers, correlating with membrane damage severity [2]
Cell Assay	- Model biological membrane photophysical assay: Model membranes (e.g., liposomes) were prepared and suspended in buffer. 3,3'-Dioctadecyloxacarbocyanine perchlorate was added at a final concentration of 1-10 μM, and the mixture was incubated at 37°C for 30 minutes. Fluorescence excitation/emission spectra, quantum yield, and fluorescence lifetime were measured using a spectrofluorometer to analyze probe-membrane interaction [3][4] - Muscle cell membrane labeling and injury assay: Cultured mouse soleus muscle cells were seeded on coverslips. 3,3'-Dioctadecyloxacarbocyanine perchlorate solution (5 μM) was added and incubated for 1 hour at 37°C to label membranes. Cells were then subjected to contraction-induced injury via electrical stimulation. Fluorescence microscopy was used to observe probe distribution (membrane vs. cytoplasm) and assess membrane integrity [2] - Neuronal DiOlistic labeling assay: Primary neuronal cultures were plated on poly-L-lysine-coated dishes. 3,3'-Dioctadecyloxacarbocyanine perchlorate-coated gold particles were delivered into cells via a gene gun. After 24-48 hours of incubation, cells were fixed, and fluorescence microscopy was performed to visualize labeled neurites and neuronal morphology [1]
Animal Protocol	- Nervous system DiOlistic labeling: Mice were anesthetized, and target neural tissues (cerebral cortex or spinal cord) were exposed. Gold particles coated with 3,3'-Dioctadecyloxacarbocyanine perchlorate (and other lipophilic dyes for multicolor labeling) were delivered into the tissues using a gene gun. Incisions were sutured, and mice were allowed to recover for 24-48 hours. Animals were then sacrificed, neural tissues were dissected, fixed, and sectioned. Fluorescence microscopy was used to image labeled neurons and neural circuits [1] - Muscle injury and membrane labeling: Mice were anesthetized, and unilateral soleus muscles were subjected to electrical stimulation to induce contraction-induced injury. 3,3'-Dioctadecyloxacarbocyanine perchlorate solution (10 μM) was topically applied to the injured muscle and incubated for 1 hour. Muscles were harvested, frozen, and sectioned. Fluorescence microscopy was used to analyze probe redistribution and evaluate membrane damage [2]
References	[1]. Multicolor "DiOlistic" labeling of the nervous system using lipophilic dye combinations. Neuron. 2000 Aug;27(2):219-25. [2]. Redistribution of cell membrane probes following contraction-induced injury of mouse soleus muscle. [3]. Photophysical studies of 3,3' dioctadecyloxacarbocyanine dye in model biological membranes and different solvents. Chem Phys Lipids. 2001 Feb;109(2):175-83. [4]. Photophysical studies of 3,3' dioctadecyloxacarbocyanine dye in model biological membranes and different solvents. Chem Phys Lipids. 2001 Feb;109(2):175-83.
Additional Infomation	- 3,3'-Dioctadecyloxacarbocyanine perchlorate is a synthetic lipophilic cationic fluorescent probe, commonly known as DiOC18(3) perchlorate [1][2][3][4] - Its core mechanism involves inserting into the lipid bilayer of biological membranes via hydrophobic and van der Waals interactions, with fluorescence properties (emission wavelength, intensity, lifetime) sensitive to the membrane microenvironment (polarity, fluidity) [3][4] - Key applications include: multicolor labeling of neurons and neural circuits in the nervous system (DiOlistic technique) [1]; assessment of plasma membrane integrity in muscle cells and other cell types [2]; photophysical studies of model biological membranes to investigate membrane structure and properties [3][4] - It is a research tool for biological imaging and membrane biology studies, not a therapeutic drug, and exhibits no pharmacological activities (e.g., anti-proliferative, anti-inflammatory) [1][2][3][4]

Solubility Data

Solubility (In Vitro)

DMF : 10 mg/mL (~11.34 mM) DMSO : ~5 mg/mL (~5.67 mM)

Solubility (In Vivo)

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. Injection Formulations (e.g. IP/IV/IM/SC) 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). 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) Oral Formulations 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). 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 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.1342 mL	5.6708 mL	11.3416 mL
	5 mM	0.2268 mL	1.1342 mL	2.2683 mL
	10 mM	0.1134 mL	0.5671 mL	1.1342 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.