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| Other Sizes |
| Targets |
C6 NBD Ceramide targets the Golgi apparatus in living and fixed cells, serving as a specific fluorescent probe for this organelle. The compound is taken up by cells and transported to the Golgi, where it can be metabolized by endogenous enzymes to fluorescent sphingomyelin and glucosylceramide. These metabolites then follow distinct intracellular trafficking pathways, allowing researchers to monitor sphingolipid transport and metabolic mechanisms. The green fluorescence (Ex=466 nm, Em=536 nm) enables visualization of Golgi morphology and changes in living cells.
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| ln Vitro |
Advice (This is our suggested protocol, which should be adjusted to suit your particular circumstances as it simply offers guidance). Golgi tagging [3]: 1. Cells on coverslips should be rinsed with HMEM before being fixed for five to ten minutes at 25°C with either glutaraldehyde or paraformaldehyde. 2. Use a balanced salt solution (HCMF, for example) to wash the fixed cells. 3. Place the culture plate with the glass coverslip in an ice-water bath and incubate it for 3 × 5 minutes (for cells that have been glutaraldehyde-fixed) using freshly made NaBH4. 4. Rinse cells multiple times (20–30 minutes) in cold HCMF, then preheat to 25°C and incubate for 60 minutes in the presence of C6 NBD-Ceramide/BSA. 5. Cleansed fixed cells with HCMF, incubate (30-90 minutes; 25°C) with 10% fetal bovine serum or 2 mg/mL BSA to improve Golgi apparatus dyeing equipment and counter-exchange excess C6 NBD-ceramide in the preparation. 6. Using a fluorescence microscope (E=536 nm, Ex=466 nm), mount the glass coverslip on the recessed slide and examine.
In vitro, C6 NBD Ceramide is used for fast and convenient green fluorescent labeling of the Golgi apparatus in living and fixed cells. The cell-permeable fluorescent ceramide analog is incorporated into the Golgi apparatus, enabling observation of Golgi morphology changes in real time. It is also metabolized to fluorescent sphingomyelin and glucosylceramide, which are then transported to other cellular compartments, allowing researchers to investigate sphingolipid transport and metabolic pathways. The green fluorescence emission at 536 nm is compatible with standard FITC filter sets on fluorescence microscopes. |
| ln Vivo |
In vivo, C6 NBD Ceramide is used primarily for cellular studies rather than whole-animal experiments. However, its ability to label the Golgi apparatus and track sphingolipid trafficking makes it valuable for understanding lipid metabolism in model organisms. For instance, it can be injected into tissues to study ceramide processing and the transport of its metabolites, such as sphingomyelin and glucosylceramide, to the plasma membrane. The green fluorescence of the NBD tag is stable under physiological conditions, enabling real-time tracking of lipid dynamics at the cellular level in living tissues.
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| Enzyme Assay |
For non-cell-based assays, C6 NBD Ceramide is used to study its interaction with lipid transport proteins such as CERT (ceramide transfer protein). A fluorescence-based lipid transfer assay is performed using a black 96-well plate. Donor vesicles containing C6 NBD Ceramide (0.5 mol%) and acceptor vesicles lacking the fluorescent lipid are mixed in assay buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA) at 37degC. The transfer of C6 NBD Ceramide from donor to acceptor vesicles is monitored by the increase in fluorescence intensity at 536 nm (Ex 466 nm). The rate of transfer is calculated to assess CERT activity or inhibitor efficacy.
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| Cell Assay |
C6 NBD Ceramide is soluble in DMSO, DMF, and ethanol. For in vitro cell staining, a 1-10 uM working solution is prepared by diluting the stock solution in PBS or serum-free culture medium. The compound is often complexed with defatted BSA (1:1 molar ratio) to enhance delivery and prevent non-specific binding. Working solutions should be prepared fresh and protected from light. For fixed cell staining, cells are first fixed with 4% paraformaldehyde, washed with PBS, and then incubated with the C6 NBD Ceramide solution for 20-60 minutes at 4degC or room temperature. For live cell labeling, cells are incubated at 37degC for 5-30 minutes. After staining, excess dye is removed by washing with PBS containing 0.2% BSA. Labeled cells are visualized using a fluorescence microscope equipped with 488 nm excitation and 520 nm emission filters. The fluorescence is well retained in fixed cells after formaldehyde fixation. C6 NBD Ceramide is stored at -20degC, protected from light.
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| Animal Protocol |
C6 NBD Ceramide is not typically administered to animals as a systemic compound. However, it can be used in ex vivo studies on isolated tissues or in cultured organ explants. For example, freshly isolated rat brain slices or liver explants are incubated in culture medium containing 5-10 uM C6 NBD Ceramide (complexed with BSA) at 37degC for 30-60 minutes. After incubation, tissues are washed, fixed with 4% paraformaldehyde, and sectioned for fluorescence microscopy analysis. This approach allows the study of ceramide processing and sphingolipid trafficking in intact tissue architecture while maintaining the advantages of fluorescence detection. The excitation/emission of 466/536 nm is suitable for confocal microscopy.
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| ADME/Pharmacokinetics |
C6 NBD Ceramide has a molecular weight of 575.74 g/mol and a molecular formula of C30H49N5O6. It is soluble in DMF (25 mg/mL), DMSO (10 mg/mL), ethanol (5 mg/mL), and DMF:PBS (pH 7.2) (1:1) at 500 ug/mL. The compound exhibits absorption maxima at 229 nm, 333 nm, and 465 nm. It appears as a crystalline solid when stored properly. For cell staining, a 5 mM stock solution in DMSO is typically prepared and stored at -20degC. The working solution is prepared by diluting the stock in PBS or serum-free medium. The compound should be protected from light during storage and handling to prevent photobleaching.
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| Toxicity/Toxicokinetics |
As a fluorescent probe, C6 NBD Ceramide is generally considered low in toxicity at the standard working concentrations of 1-10 uM. No significant cytotoxic effects are observed during typical staining incubations of 15-60 minutes. However, the compound is for research use only and is not intended for human or veterinary use. Standard laboratory safety precautions should be followed when handling this compound. Avoid prolonged exposure to light to prevent photobleaching. The product is not classified as a carcinogen or reproductive hazard based on available information.
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| References | |
| Additional Infomation |
N-{6-[(7-nitro-2,1,3-benzodiazol-4-yl)amino]hexanoyl}sphingosine is an N-acylsphingosine in which the amino nitrogen is converted to a 6-{[N-(7-nitrobenzodiazol-2,1,3-oxadiazol-4-yl)amino]}hexanoamide group. It can be used as a fluorescent probe.
C6 NBD Ceramide is a widely used fluorescent probe for studying the Golgi apparatus, sphingolipid transport, and ceramide metabolism in live and fixed cells. The NBD fluorophore is environmentally sensitive, exhibiting increased fluorescence in hydrophobic environments such as cellular membranes. This probe is often used in combination with other fluorescent markers for multicolor imaging studies. The compound is metabolized to NBD-sphingomyelin and NBD-glucosylceramide, allowing researchers to track the conversion of ceramide to more complex sphingolipids. It is not a drug and has no clinical approval status. The product is supplied for research use only with a purity of ≥98%. |
| Molecular Formula |
C30H49N5O6
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|---|---|
| Molecular Weight |
575.73996
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| Exact Mass |
575.368
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| CAS # |
94885-02-6
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| PubChem CID |
16219783
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| Appearance |
Brown to orange solid powder
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| Density |
1.2±0.1 g/cm3
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| Index of Refraction |
1.562
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| LogP |
8.25
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
23
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| Heavy Atom Count |
41
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| Complexity |
737
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| Defined Atom Stereocenter Count |
2
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| SMILES |
CCCCCCCCCCCCC/C=C/[C@H]([C@H](CO)NC(=O)CCCCCNC1=CC=C(C2=NON=C12)[N+](=O)[O-])O
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| InChi Key |
HZIRBXILQRLFIK-VPZZKNKNSA-N
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| InChi Code |
InChI=1S/C30H49N5O6/c1-2-3-4-5-6-7-8-9-10-11-12-13-15-18-27(37)25(23-36)32-28(38)19-16-14-17-22-31-24-20-21-26(35(39)40)30-29(24)33-41-34-30/h15,18,20-21,25,27,31,36-37H,2-14,16-17,19,22-23H2,1H3,(H,32,38)/b18-15+/t25-,27+/m0/s1
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| Chemical Name |
N-[(E,2S,3R)-1,3-dihydroxyoctadec-4-en-2-yl]-6-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]hexanamide
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
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
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| 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
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] 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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7369 mL | 8.6845 mL | 17.3690 mL | |
| 5 mM | 0.3474 mL | 1.7369 mL | 3.4738 mL | |
| 10 mM | 0.1737 mL | 0.8684 mL | 1.7369 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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