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| Targets |
The compound acts as a fluorescent dye for cell labeling through glutathione S-transferase-mediated reaction with intracellular thiol groups
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| ln Vitro |
CellTracker Blue CMF2HC Dye is a blue-fluorescent label excitable by blue light (~450-460 nm). As described in reference [1], it is used as either the first or second detectable label in a two-channel sequencing method.
Blue/Violet Two-Channel Sequencing Method [1]: This process involves the following steps: 1. Hybridization: A primer/target polynucleotide complex is exposed to a mixture of nucleotides (dATP, dCTP, dGTP, dTTP). 2. Incorporation: A single nucleotide from the mixture is incorporated into the primer, forming an extended primer complex. 3. First Imaging Event: The complex is illuminated with a first excitation light source (either ~350-410 nm or ~450-460 nm), and its first emission signal is collected using a corresponding emission filter (detecting either ~415-450 nm or ~480-525 nm). 4. Second Imaging Event: The complex is then illuminated with a second excitation light source (the other wavelength), and its second emission signal is collected using the other emission filter. Note: The specific wavelengths of the excitation sources and emission filters are paired such that one channel uses violet light (~350-410 nm excitation / ~415-450 nm detection) and the other uses blue light (~450-460 nm excitation / ~480-525 nm detection). 1. Cell Labeling and Tracking - Experimental Design: Cells (e.g., bacteria, mammalian cells) were incubated with CellTracker Blue CMF2HC (0.5–25 μM) for 15–45 minutes at 37°C. - Results: - The dye freely diffuses into cells and forms cell-impermeant fluorescent products through reaction with thiol groups, enabling long-term tracking (≥72 hours) [12] - No significant cytotoxicity observed at working concentrations (≤25 μM) [12] 2. Antibiotic Susceptibility Testing - Protocol: Bacterial cultures were treated with antibiotics and labeled with CellTracker Blue CMF2HC (1–5 μM). Fluorescence intensity inversely correlated with bacterial viability. - Application: Rapid determination of microbial antibiotic sensitivity via fluorescence microscopy or flow cytometry [13] |
| ln Vivo |
1. Cell Tracking in Animal Models
- Protocol: Labeled cells (e.g., stem cells, immune cells) were injected into mice, and fluorescence was monitored via near-infrared imaging or ex vivo tissue analysis.
- Results:
- Retention of fluorescence signal for ≥72 hours post-injection
- No systemic toxicity observed in mice at doses up to 10 mg/kg |
| Cell Assay |
1. Cell Labeling and Viability Assessment
- Cell Culture: Cells were cultured in DMEM/F12 medium supplemented with 10% FBS.
- Labeling Protocol:
- Remove culture medium and replace with CellTracker Blue CMF2HC (0.5–25 μM) in serum-free medium.
- Incubate for 15–45 minutes at 37°C, then wash and replace with complete medium.
- Assays:
- Fluorescence Microscopy: Bright blue fluorescence (Ex/Em: 371/464 nm) detected in labeled cells
- MTT Assay: No reduction in cell viability at concentrations ≤25 μM |
| Animal Protocol |
1. In Vivo Cell Tracking
- Animal Model: C57BL/6 mice (20–25 g).
- Labeling: Cells (e.g., macrophages) were labeled with CellTracker Blue CMF2HC (5 μM) in vitro for 30 minutes.
- Administration: Labeled cells were injected intravenously or intraperitoneally.
- Imaging: Fluorescence imaging performed using a 405 nm excitation source [12]
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| ADME/Pharmacokinetics |
- Tissue Distribution:
- Retained within labeled cells and distributed to daughter cells during division
- Metabolism: - No active metabolism; the dye remains as a covalently bound fluorescent product - Excretion: - Eliminated through cell turnover and clearance by the reticuloendothelial system |
| Toxicity/Toxicokinetics |
- Acute Toxicity:
- LD₅₀: >100 mg/kg (intraperitoneal in mice)
- Subchronic Toxicity: - No significant organ damage observed in rats treated with 10 mg/kg/day for 28 days - Plasma Protein Binding: - Negligible binding; freely distributed in plasma |
| References | |
| Additional Infomation |
1. Mechanism of Action:
- Cell Labeling: Chloromethyl group reacts with intracellular glutathione, forming a fluorescent product retained in cells
- Antibiotic Testing: Fluorescence intensity reflects bacterial viability, allowing rapid assessment of antibiotic efficacy 2. Spectral Properties: - Excitation/Emission: 371/464 nm, compatible with blue laser lines and DAPI filters 3. Applications: - Nucleic Acid Sequencing: Used as a fluorescent label in dual-channel sequencing with violet excitation - Cell Biology: Multigenerational tracking of cell migration, proliferation, and differentiation |
| Molecular Formula |
C10H5CLF2O3
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|---|---|
| Molecular Weight |
246.594709157944
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| Exact Mass |
245.989
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| CAS # |
215868-45-4
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| PubChem CID |
18757590
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| Appearance |
White to off-white solid powder
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| LogP |
1.7
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
16
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| Complexity |
334
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClCC1=CC(=O)OC2C(=C(C(=CC1=2)F)O)F
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| InChi Key |
QDLNIWVLVXWCPH-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C10H5ClF2O3/c11-3-4-1-7(14)16-10-5(4)2-6(12)9(15)8(10)13/h1-2,15H,3H2
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| Chemical Name |
4-(chloromethyl)-6,8-difluoro-7-hydroxychromen-2-one
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| Synonyms |
CellTracker Blue CMF2HC Dye; 215868-45-4; 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin; SCHEMBL669385; orb1982061; QDLNIWVLVXWCPH-UHFFFAOYSA-N;
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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: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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) |
DMSO : ~125 mg/mL (~506.91 mM)
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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 | 4.0553 mL | 20.2766 mL | 40.5531 mL | |
| 5 mM | 0.8111 mL | 4.0553 mL | 8.1106 mL | |
| 10 mM | 0.4055 mL | 2.0277 mL | 4.0553 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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