Research has confirmed that iron is the most abundant transition metal element in living organisms. It participates in various physiological activities. In recent years, intracellular free iron ions have received increasing attention due to their high reactivity and association with cell damage and death. Free iron ions exist in the form of stable Fe2+and Fe3+within cells. Considering the reducing environment within cells, metal transporters, and the water solubility of Fe2+, it is believed that revealing the behavior of Fe2+within cells is more important than Fe3+. Mito FerroGreen is a novel fluorescent probe used to detect the ferrous ion Fe2+in mitochondria (the site of iron sulfur cluster and heme protein synthesis). The increase in fluorescence intensity after the reaction between Mito FerroGreen and Fe2+is irreversible, which is different from fluorescence probes such as Fluo-3 that can monitor calcium ions in real time.
Physicochemical Properties
| Appearance | Solid powder |
| Synonyms | Mitochondrial iron probe |
| 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: 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) |
Biological Activity
| Targets | Mitochondria |
| ln Vitro | As one of the cornerstones of clinical cardiovascular disease treatment, statins have an extensive range of applications. However, statins commonly used have side reactions, especially muscle-related symptoms (SAMS), such as muscle weakness, pain, cramps, and severe condition of rhabdomyolysis. This undesirable muscular effect is one of the chief reasons for statin non-adherence and/or discontinuation, contributing to adverse cardiovascular outcomes. Moreover, the underlying mechanism of muscle cell damage is still unclear. Here, we discovered that ferroptosis, a programmed iron-dependent cell death, serves as a mechanism in statin-induced myopathy. Among four candidates including atorvastatin, lovastatin, rosuvastatin, and pravastatin, only atorvastatin could lead to ferroptosis in human cardiomyocytes (HCM) and murine skeletal muscle cells (C2C12), instead of human umbilical vein endothelial cell (HUVEC). Atorvastatin inhibits HCM and C2C12 cell viability in a dose-dependent manner, accompanying with significant augmentation in intracellular iron ions, reactive oxygen species (ROS), and lipid peroxidation. A noteworthy investigation found that those alterations particularly occurred in mitochondria and resulted in mitochondrial dysfunction. Biomarkers of myocardial injury increase significantly during atorvastatin intervention. However, all of the aforementioned enhancement could be restrained by ferroptosis inhibitors. Mechanistically, GSH depletion and the decrease in nuclear factor erythroid 2-related factor 2 (Nrf2), glutathione peroxidase 4 (GPx4), and xCT cystine-glutamate antiporter (the main component is SLC7A11) are involved in atorvastatin-induced muscular cell ferroptosis and damage. The downregulation of GPx4 in mitochondria-mediated ferroptosis signaling may be the core of it. In conclusion, our findings explore an innovative underlying pathophysiological mechanism of atorvastatin-induced myopathy and highlight that targeting ferroptosis serves as a protective strategy for clinical application [1]. |
| Enzyme Assay |
Staining example:< br>
1. Mitochondrial localization To confirm whether Mito FerroGreen can specifically localize within mitochondria, staining was performed together with mitochondrial staining reagent (MitoBright Deep Red ※), and the experimental results confirmed that Mito FerroGreen selectively stains within mitochondria. Add 5 μ mol/l Mito FerroGreen and 200 nmol/l MitoBright Deep Red mitochondrial staining probe to HeLa cells, and culture in a CO2 incubator for 30 minutes. Then add 100 μ mol/l ammonium iron sulfate (II) and culture the mixed cell solution in the CO2 incubator for 1 hour before observing fluorescence. Mito-FerroGreen Excitation wavelength: 488 nm Emission wavelength: 500-565 nm MitoBright Deep Red Excitation wavelength: 640 nm Emission wavelength: 656-700 nm 2. Fluorescence imaging of iron ions within mitochondria Inoculate HeLa cells into MEM medium containing serum and add Mito FerroGreen to detect divalent iron in the mitochondria of HeLa cells by fluorescence detection. In HeLa cells with added iron ions, significant enhancement of Mito FerroGreen fluorescence was observed. In cells treated with iron chelators, almost no fluorescence of Mito FerroGreen was observed. In this way, it was confirmed that the differences in iron content and fluorescence intensity in mitochondria are correlated. 3. High selectivity and high signal for divalent iron ions Add 2 μ l of 1mol/l Mito FroGreen, 2 μ l of 10mmol/l of various metals, and 20 μ l of 1mg/ml esterifying enzyme to 1ml of 50mmol/l HEPES Buffer (pH 7.4), and measure the fluorescence intensity after 1 hour of reaction at room temperature. Excitation wavelength: 500 nm Emission wavelength: 535 nm 4. Suitable for general filters The excitation wavelength of Mito FerroGreen is 488nm, and the maximum excitation wavelength can reach 505nm< br> Add 6 μ l of 1mol/l Mito FroGreen, 6 μ l of 10mmol/l ammonium iron (II) sulfate, and 20 μ l of 1mg/ml esterifying enzyme to 3ml of 50 mmol/l HEPES Buffer (pH 7.4). Measure the fluorescence intensity after 1 hour of reaction at 37 ℃. Excitation wavelength: 500 nm Emission wavelength: 535 nm |
| Cell Assay | FerroOrange and Mito-FerroGreen probes were employed to assess intracellular and mitochondrial iron content, respectively. HCM and C2C12 cells were seeded into 24-well plates and exposed to atorvastatin at the concentrations of 40 uM alone, or other treatments for 24 h. After that, the cells were stained with FerroOrange (1 μM) or Mito-FerroGreen (5 μM) probes along with Hoechst 33,342 (5 μg/ml). After 30 min of lucifuge incubation, the cells were then evaluated by a Leica SP8 confocal fluorescence microscope [1]. |
| References |
[1]. Atorvastatin Induces Mitochondria-Dependent Ferroptosis via the Modulation of Nrf2-xCT/GPx4 Axis. Front Cell Dev Biol. 2022 Mar 3:10:806081. |
Solubility Data
| Solubility (In Vitro) | This product is soluble in acetonitrile, methanol, and dimethyl alcohol. |
| 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.) |