| Size | Price | |
|---|---|---|
| 500mg | ||
| 1g | ||
| Other Sizes |
| Targets |
Mitochondrial membrane potential (mtMP); TMRM is a fluorescent probe that accumulates in mitochondria in response to negative membrane potential. [2][3]
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|---|---|
| ln Vitro |
1. TMRM working solution components 1.1 Set up the stock solution. To get a 5 mM stock solution, mix 1 milligram of TMRM with 525 μL of DMSO. 1.2 Get the solution for storage ready. Pour the storage solution into a 1–20 μM TMRM working solution along with pre-made bone marrow cell-free or PBS. Note: Please prepare the TMRM working fluid for usage by adjusting its concentration to suit the current circumstances. 2. Cell staining (suspended cells) 2.1 Centrifuge the cells, add PBS, and wash twice, ensuring that each wash has a cell density of 5 × 106/mL. 2.2 After adding 1 mL of the TMRM working solution, give it 30 to 60 minutes. 2.3 Centrifuge for 3–4 minutes at 400 g, then remove the supernatant. 2.4 Wash the cells twice with PBS, giving them five minutes each time. 2.5 Use a fluorescence microscope or flow cytometer to observe the cells after resuspending them in 1 mL of serum-free cells or PBS. 3. Aspirate a tiny amount of adherent cells by removing the coverslips from the culture medium (step 3.2). 3.1 Culture adherent cells on sterile coverslips. 3.3 Add 100 μL of the dye working solution, gently shake to coat the cells, and let sit for five to thirty minutes. 3.4 Use a fluorescence microscope or flow cytometer to observe after aspirating the dye working solution and washing the sample two to three times in culture media for five minutes each time.
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| Enzyme Assay |
- Oxygen consumption rate (OCR) measurement: Isolated mitochondria or tissue homogenates were resuspended in KCl-enriched buffer (80 mM KCl, 10 mM Tris/HCl, 3 mM MgCl₂, 1 mM EDTA, 5 mM potassium phosphate, pH 7.4). Substrates (glutamate, pyruvate, malate, succinate), ADP, oligomycin, FCCP, rotenone, and antimycin A were added sequentially. OCR was measured at 37°C using an Oroboros Oxygraph-2K with fluorescence LED2 module. [2]
- Mitochondrial membrane potential (mtMP) measurement: Safranin (2.5 μM) or TMRM (2 μM) was used as fluorescent probe. The Oroboros system simultaneously recorded fluorescence signals (excitation/emission: safranin 495/587 nm; TMRM 530/592 nm) and OCR. [2] - 1P ratiometric imaging: Cells were excited sequentially at 546 and 573 nm (5 nm bandwidth) using a monochromator. Emission was detected at 620/60 nm. Ratio images (573/546) were formed every 200 msec. [3] - Fluorescence lifetime imaging (FLIM): Two-photon excitation at 810 nm was used. Phase and modulation images were collected using a FastFLIM module. Fluorescence lifetimes were analyzed using the phasor plot method. [3] |
| Cell Assay |
- Isolated rat cortical mitochondria: Mitochondria were isolated from rat brain cortex by differential centrifugation. Protein content was measured by Bradford assay. For OCR/mtMP measurements, 200–300 μg of mitochondrial protein was used. [2]
- Primary rat hippocampal neuron cultures: Cultures were prepared from embryonic day 18 rat hippocampi and grown on glial feeder layers for 12–30 days in vitro. TMRM was bath-applied at concentrations of 50 nM to 25 μM. Imaging was performed at room temperature. [3] - Apoptosis assay: Cultures were exposed to TMRM for 1 hr at 37°C, then post-incubated for 18 hrs. Cells were stained with Hoechst 33258 (2 μg/mL) for 20 min. Apoptotic cells were identified as brightly fluorescent nuclei under UV excitation. Cell survivability was calculated as percentage of live cells. [3] - Calcium imaging: Cells were loaded with Fluo-3 AM (5 μM) for 1 hr at 37°C, then post-incubated for 1 hr. Dual-probe imaging of Fluo-3 and TMRM was performed using two-photon microscopy. [3] |
| Toxicity/Toxicokinetics |
- In isolated rat cortical mitochondria, TMRM (2 μM) decreased coupled respiration by approximately 27% compared to dye-free controls, indicating mild toxicity. Maximal uncoupled respiration was not affected. [2]
- In cultured hippocampal neurons, exposure to TMRM (50–200 nM) for 1 hr did not significantly affect cell survivability (∼80%, similar to controls). Higher concentrations (0.5 μM and 2.5 μM) induced substantially greater cell death. A 1-hr exposure to 2.5 μM TMRM resulted in approximately 40% survivability. Survivability decreased progressively over 48 hrs following a 1-hr exposure, consistent with apoptosis. Shorter exposures (5–15 min) were less toxic. [3] - Prolonged exposure to high concentrations of TMRM (1–25 μM) induced spontaneous increases in fluorescence, oscillations, and waves in some neurons, which were followed by apoptosis. [3] |
| References |
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| Additional Infomation |
- TMRM (tetramethylrhodamine methyl ester) is a cationic, membrane-permeable fluorescent dye that accumulates in mitochondria in response to negative mitochondrial membrane potential (Nernstian distribution). It is widely used to study mitochondrial function in living cells. [2][3]
- TMRM fluorescence is quenched when the dye accumulates in mitochondrial membranes. Unquenched dye partitions in the mitochondrial intermembranous space. Ratiometric imaging (573/546 nm excitation) can distinguish between quenched and unquenched dye. [3] - The two-photon excitation peak of TMRM is 830 nm. In this study, 820 nm was used to minimize phototoxicity. [3] - TMRM is amphipathic and readily partitions into cellular membranes, including nuclear membranes. [3] - Compared to safranin, TMRM showed less toxicity on FCCP-stimulated maximal respiration, making it a better choice for simultaneous OCR and mtMP measurements. However, safranin exhibited higher sensitivity to changes in mtMP. [2] |
| Molecular Formula |
C25H25N2O3
|
|---|---|
| Molecular Weight |
401.48
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| Exact Mass |
401.186
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| CAS # |
115532-49-5
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| Related CAS # |
TMRM Perchlorate;115532-50-8
|
| PubChem CID |
5009757
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| Appearance |
Typically exists as solid at room temperature
|
| LogP |
3.3
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
|
| Heavy Atom Count |
30
|
| Complexity |
772
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
CN(C)C1=CC2=C(C=C1)C(=C3C=CC(=[N+](C)C)C=C3O2)C4=CC=CC=C4C(=O)OC
|
| InChi Key |
WAWRKBQQBUDAMY-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H25N2O3/c1-26(2)16-10-12-20-22(14-16)30-23-15-17(27(3)4)11-13-21(23)24(20)18-8-6-7-9-19(18)25(28)29-5/h6-15H,1-5H3/q+1
|
| Chemical Name |
[6-(dimethylamino)-9-(2-methoxycarbonylphenyl)xanthen-3-ylidene]-dimethylazanium
|
| Synonyms |
TMRM; 115532-49-5; [6-(dimethylamino)-9-(2-methoxycarbonylphenyl)xanthen-3-ylidene]-dimethylazanium; 3,6-Bis(dimethylamino)-9-[2-(methoxycarbonyl)phenyl]xanthylium; 3,6-Bis(dimethylamino)-9-(2-(methoxycarbonyl)phenyl)xanthylium;
|
| 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 |
| 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 | 2.4908 mL | 12.4539 mL | 24.9078 mL | |
| 5 mM | 0.4982 mL | 2.4908 mL | 4.9816 mL | |
| 10 mM | 0.2491 mL | 1.2454 mL | 2.4908 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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