RhBIV
RhBIV is a thermosensitive rhodamine B (RhB)-derived fluorescent probe that fluorescently labels cellular mitochondria and detects mitochondrial temperature in vitro and in vivo. RhBIV contains a quaternized rhodamine B core, which confers a positive charge for binding to the negatively charged mitochondrial membrane, and an ortho-substituted electron-donating piperidine group; at lower temperatures, RhBIV forms an unstable ion pair form under kinetic control and exhibits strong fluorescence, whereas elevated temperature disrupts or partially interrupts the ion pair form, favoring formation of the conjugated RhBV form and leading to loss of fluorescence. RhBIV exhibits decreased fluorescence responsiveness with increasing temperature over the range of 20-42°C. RhBIV enables biological imaging of cells and mice, mitochondrial thermogenesis imaging, dynamic observation in mitochondrial thermogenesis imaging experiments, and non-invasive imaging of mitochondrial function in living animals (Ex/Em = 520/592 nm).
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- CAS No.: 2713577-11-6
- 화학식: C33H42ClN3O
- 분자량:532.16
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보관:
Please store the product under the recommended conditions in the Certificate of Analysis.
To place orders, for customer services and technical support, please contact: MedChemExpress USA
Tel: 609-228-6898 E-mail: [email protected] [email protected]
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Biological Activity
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Chemical Information
- Protocol
- 순도&문서
- References
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Help & FAQs
Biological Activity
제품 설명
In Vitro
Guide (The following is our recommended protocol. This protocol is for reference only and should be adjusted according to your specific needs).
2. Working Solution Preparation
Working concentration: 1 μM RhBIV, used for fluorescent labeling of cell mitochondria.
3. Staining Procedure
3.1 For live cells[1][2]:
3.1.1 Use RhBIV working solution for fluorescent labeling of cell mitochondria.
3.1.2 For temperature-response measurements, test within the range of 20-42 °C; when the temperature increases from 27 °C to 41 °C, the fluorescence intensity decreases.
3.2 For HepG2 cells[3]:
3.2.1 Culture HepG2 cells in DMEM containing 5% CO2 at 37°C.
3.2.2 Incubate with 3 and 10 μM CAT or CA at 37°C for 6 h; incubate the CCCP (HY-100941) group for 30 min to induce mitochondrial thermogenic response.
3.2.3 Use the RhBIV probe to detect mitochondrial temperature in cells.
4. Controls
4.1 Cell-based evaluation: control group (Con) and CCCP-induced mitochondrial thermogenesis model group (Mod); the CCCP group is incubated for 30 min to induce mitochondrial thermogenic response.
5. Detection and Analysis
5.1 Instrument type: microplate reader with temperature control and full-wavelength scanning; laser confocal microscope; fluorescence imaging/microscopy.
5.2 Wavelength settings: RhBIV excitation at 520 nm, emission at 592 nm.
5.3 Result analysis:
5.3.1 RhBIV emits no background signal at 520 nm excitation, appearing pseudo-green; its signal partially overlaps with MitoTracker, appearing orange, and is stronger in the cytoplasmic mitochondrial membrane, with an MCC of approximately 0.96.
5.3.2 When the temperature increases from 27 °C to 41 °C, the fluorescence intensity of RhBIV decreases by 55%.
5.3.3 In the cell-based evaluation, the CCCP-induced mitochondrial thermogenesis model group (Mod) shows a significantly reduced RhBIV fluorescence response compared with the control group (Con). In cells, CCCP causes a decrease in fluorescence intensity in the CCCP group; CA (10 μM) significantly enhances the fluorescence response, whereas the CAT groups (3 μM and 10 μM) show no significant change.
MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only. Further protocols information, click here.
In Vivo
1. In vivo imaging in mice[1][2][3]:
Mice are randomly divided into five groups and injected with RhBIV (10 mg/kg, i.v.).
1. Inject 10 mg/kg RhBIV via tail vein.
2. In the aging model, inject RhBIV intravenously and measure the fluorescence response after 1.5 h.
3. For organ distribution, harvest organs 1 h after injection.
4. Real-time fluorescence scanning can be performed within 60-150 min.
5. For mitochondrial thermogenesis imaging of mouse liver, use 10 mg/kg RhBIV probe.
2. Controls
2.1 Cell-based evaluation: control group (Con) and CCCP-induced mitochondrial thermogenesis model group (Mod); the CCCP group is incubated for 30 min to induce mitochondrial thermogenesis.
2.2 In vivo antipyretic evaluation: control group (Con) and 2,4-dinitrophenol-induced fever group (Mod, 28 mg/kg).
2.3 Aging evaluation: control group and D-galactose-induced aging model group.
2.4 In vivo control: the control group is administered normal saline by gavage.
3. Detection and analysis
3.1 Instrument type: small animal in vivo imaging system for dynamic fluorescence imaging.
3.2 Wavelength settings: excitation 530 nm, emission 600 nm.
3.3 Result analysis:
3.3.1 In mice, tail vein injection of 10 mg/kg allows RhBIV to distribute throughout the body and increase over time; 1 h after injection, RhBIV is mainly enriched in the liver, with minimal enrichment in kidney and lymph node tissues. Real-time fluorescence scanning shows that RhBIV fluorescence intensity is relatively stable within 60-150 min, reaches Cmax within 90 min, and t1/2 is approximately 9.6 h. The RhBIV probe is used to evaluate mitochondrial thermogenesis in mouse liver.
3.3.2 In the antipyretic experiment, the 2,4-dinitrophenol-induced fever group (Mod, 28 mg/kg) shows a low fluorescence response compared with the control group (Con); in vivo, compared with the model group, the fluorescence intensity in the ASP (115 mg/kg) and CAT (130 mg/kg) groups increases significantly, while CAT (43 mg/kg) shows no significant difference.
3.3.3 In the aging model, the D-galactose-induced aging mouse model shows a stronger fluorescence response compared with the control group; administration of VB3 and G-CK alleviates mitochondrial damage in aging mice.
3.3.4 The RhBIV probe responds to body temperature.
MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.
Chemical Information
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CAS No. 2713577-11-6
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분자량 532.16
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화학식 C33H42ClN3O
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SMILES
CCN(C1=CC2=C(C(C3=C(C=CC=C3)CN4CCCCC4)=C5C=C/C(C=C5O2)=[N+](CC)\CC)C=C1)CC.[Cl-]
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선적
Room temperature in continental US; may vary elsewhere.
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보관
Please store the product under the recommended conditions in the Certificate of Analysis.
Protocol
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Mitochondrial membrane-potential fluorescent assay
Mitochondrial membrane potential fluorescent assays estimate ΔΨm in living cells using lipophilic cationic dyes such as TMRM, TMRE, rhodamine 123, and JC-1, which accumulate in mitochondria according to membrane polarization; loss of signal after FCCP or CCCP treatment is interpreted as mitochondrial depolarization. TMRM/TMRE and rhodamine 123 are commonly used for semi-quantitative live-cell microscopy or flow cytometry, while JC-1 can report a shift from red aggregate fluorescence to green monomer fluorescence during depolarization; interpretation requires controls because dye concentration, quenching mode, cell type, dye efflux, and mitochondrial mass can affect fluorescence independently of ΔΨm.
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Mitochondrial membrane-potential and mitochondrial mass staining
Mitochondrial membrane potential staining measures the electrochemical polarization across the mitochondrial inner membrane in live cells using lipophilic cationic fluorescent probes; early rhodamine-based work showed that selective mitochondrial dye accumulation is lost when the mitochondrial transmembrane potential is dissipated. JC-1 reports mitochondrial polarization by shifting from green monomer fluorescence to red J-aggregate fluorescence as dye concentration increases within energized mitochondria; therefore, the red/green fluorescence ratio is used as a relative readout of mitochondrial membrane potential. TMRE or TMRM staining provides a single-channel relative readout because these cationic rhodamine esters accumulate in polarized mitochondria, and lower fluorescence indicates reduced mitochondrial polarization when acquisition and dye-loading conditions are controlled. Mitochondrial mass staining is commonly performed with MitoTracker Green FM or related MitoTracker dyes as
순도&문서
References
[1]. Shen F, et al. Small-Molecule Fluorogenic Probe for the Detection of Mitochondrial Temperature . Analytical chemistry. 2021 Oct 12;93(40):13417-13420. [Content Brief]
[2]. Sun Y, et al. Recent progress on small molecular temperature-sensitive fluorescent probes. Biotechnology and bioengineering. 2023 Jan;120(1):7-21. [Content Brief]
[3]. Shen F, et al. Catalpolaglycone disrupts mitochondrial thermogenesis by specifically binding to a conserved lysine residue of UCP2 on the proton leak tunnel. Phytomedicine : international journal of phytotherapy and phytopharmacology. 2024 Mar;125:155356. [Content Brief]
Calculators
Concentration (start) × Volume (start) = Concentration (final) × Volume (final)
RhBIV Related Classifications
HY-D3144 Related Classifications
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