Rhodamine 123 (chloride) (Synonyms: R-22420,R-302,RH123) |
| Catalog No.GC15795 |
Rhodamine 123 (chloride) is a cell-permeable cationic fluorescent probe that specifically recognizes mitochondrial membrane potential, with maximum excitation/emission light of 507/529nm.
Products are for research use only. Not for human use. We do not sell to patients.
Cas No.: 62669-70-9
Sample solution is provided at 25 µL, 10mM.
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Rhodamine 123 (chloride) is a cell-permeable cationic fluorescent probe that specifically recognizes mitochondrial membrane potential, with maximum excitation/emission light of 507/529nm[1]. Rhodamine 123 is a highly efficient mitochondrial fluorescent dye, mainly used to study mitochondrial function, cell viability and multidrug resistance (MDR) detection[2]. Rhodamine 123 can selectively accumulate in the mitochondria of living cells. When the mitochondrial membrane potential is normal, its fluorescence intensity is positively correlated with the membrane potential. When the mitochondria are depolarized, the dye is released into the cytoplasm, resulting in a decrease in fluorescence[3]. Rhodamine 123 is a typical ABC transporter substrate, especially P-glycoprotein (P-gp). In multidrug-resistant tumor cells, due to the active P-gp, these cells will excrete Rhodamine 123, resulting in a decrease in intracellular fluorescence intensity[4]. At a certain concentration, rhodamine dyes have low toxicity to cells, so they are often used to detect mitochondria in animal cells, plant cells and microorganisms[5].
References:
[1]Casto D, Le T, Glaser D D. RE: Rhodamine-123: A Novel Cationic Mitochondrial Probe[J]. 2011.
[2]Reungpatthanaphong P, Dechsupa S, Meesungnoen J, et al. Rhodamine B as a mitochondrial probe for measurement and monitoring of mitochondrial membrane potential in drug-sensitive and-resistant cells[J]. Journal of biochemical and biophysical methods, 2003, 57(1): 1-16.
[3]Shapiro H M. Membrane potential estimation by flow cytometry[J]. Methods, 2000, 21(3): 271-279.
[4]Ludescher C, Thaler J, Drach D, et al. Detection of activity of P‐glycoprotein in human tumour samples using rhodamine 123[J]. British journal of haematology, 1992, 82(1): 161-168.
[5]Zeng S, Liu X, Kafuti Y S, et al. Fluorescent dyes based on rhodamine derivatives for bioimaging and therapeutics: recent progress, challenges, and prospects[J]. Chemical Society Reviews, 2023.
This plan only provides a guide, please modify it to meet your specific needs.
1. Solution preparation
(1) Stock solution: Dissolve Rhodamine 123 in DMSO to prepare a stock solution with a concentration of 5mM.
Note: Store unused stock solution in aliquots at -20°C or -80°C away from light and avoid repeated freezing and thawing.
(2) Working solution: Dilute the stock solution with a suitable buffer (such as serum-free culture medium, HBSS or PBS) to prepare a Rhodamine 123 working solution with a concentration of 1-20μM.
Note: Please adjust the optimal working concentration according to the actual situation or refer to the literature to set the gradient concentration by yourself. The working solution must be prepared and used immediately.
2. Cell suspension staining (taking 6-well plate as an example)
(1) Suspended cells are centrifuged at 1000g for 3-5min. Discard the supernatant and wash twice with PBS, each time for 5min.
(2) Adherent cells are washed twice with PBS, trypsin is added to digest the cells, and after digestion, they are centrifuged at 1000g for 3-5min.
(3) Add 1mL Rhodamine 123 working solution to resuspend the cells and incubate at room temperature in the dark for 5-30 minutes. The optimal culture time varies for different cells.
(4) After incubation, centrifuge at 1000g for 5 minutes, remove the supernatant, and wash with PBS 2-3 times, 5 minutes each time.
(5) Resuspend the cells in serum-free cell culture medium or PBS and observe by fluorescence microscopy or flow cytometry.
3. Cell adhesion staining
(1) Culture adherent cells on sterile coverslips.
(2) Remove the coverslip from the culture medium, aspirate the excess culture medium, and place the coverslip in a humid environment.
(3) Add 100μL Rhodamine 123 working solution from one corner of the coverslip, gently shake to evenly cover all cells with the dye, and incubate at room temperature in the dark for 30-60 minutes.
(4) Aspirate the dye working solution and wash the coverslip 2-3 times with culture medium, 5 minutes each time. Observe by fluorescence microscopy.
4. Experimental steps for measuring mitochondrial membrane potential using Rhodamine 123[1] (Source literature, for reference only)
(1) After 24 hours of cell culture, add the specified concentration of methylmercuric chloride and/or pyruvate, and then incubate for another 3 hours.
(2) Wash the cells with HBSS buffer to remove unbound reagents.
(3) Add 5μM Rhodamine 123 working solution (dissolved in HBSS buffer) to the cells and incubate at room temperature in the dark for 30 minutes.
(4) Collect the incubated cells with 600μL ice-cold HBSS buffer.
(5) Transfer 100μL of cell suspension to half of the wells of a 96-well plate and measure the fluorescence intensity with an excitation wavelength of 488nm and an emission wavelength of 530nm.
(6) Count the cells in 300μL of cell suspension to standardize the mitochondrial membrane potential.
Note: All fluorescent dyes have quenching problems. Please try to avoid light to slow down fluorescence quenching. For your safety and health, please wear a lab coat and disposable gloves when operating.
References:
[1] Lee J Y, Ishida Y, Takahashi T, et al. Transport of pyruvate into mitochondria is involved in methylmercury toxicity[J]. Scientific reports, 2016, 6(1): 21528.
| Cas No. | 62669-70-9 | SDF | |
| Synonyms | R-22420,R-302,RH123 | ||
| Chemical Name | 3,6-diamino-9-[2-(methoxycarbonyl)phenyl]-xanthylium, monochloride | ||
| Canonical SMILES | NC1=CC2=[O+]C3=C(C(C4=CC=CC=C4C(OC)=O)=C2C=C1)C=CC(N)=C3.[Cl-] | ||
| Formula | C21H17N2O3 • Cl | M.Wt | 380.8 |
| Solubility | ≤10mg/ml in ethanol;10mg/ml in DMSO;10mg/ml in dimethyl formamide;10mg/ml in HCl(0.1M) | Storage | 4°C, protect from light |
| General tips | Please select the appropriate solvent to prepare the stock solution according to the
solubility of the product in different solvents; once the solution is prepared, please store it in
separate packages to avoid product failure caused by repeated freezing and thawing.Storage method
and period of the stock solution: When stored at -80°C, please use it within 6 months; when stored
at -20°C, please use it within 1 month. To increase solubility, heat the tube to 37°C and then oscillate in an ultrasonic bath for some time. |
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| Shipping Condition | Evaluation sample solution: shipped with blue ice. All other sizes available: with RT, or with Blue Ice upon request. | ||
| Prepare stock solution | |||
|
1 mg | 5 mg | 10 mg |
| 1 mM | 2.6261 mL | 13.1303 mL | 26.2605 mL |
| 5 mM | 0.5252 mL | 2.6261 mL | 5.2521 mL |
| 10 mM | 0.2626 mL | 1.313 mL | 2.6261 mL |
Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)
g
μL
Step 2: Enter the in vivo formulation (This is only the calculator, not formulation. Please contact us first if there is no in vivo formulation at the solubility Section.)
Calculation results:
Working concentration: mg/ml;
Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid 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 master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O, mix and clarify.
Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.
Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such as vortex, ultrasound or hot water bath can be used to aid dissolving.
3. All of the above co-solvents are available for purchase on the GlpBio website.
Quality Control & SDS
- View current batch:
- Purity: >99.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
Average Rating: 5 (Based on Reviews and 37 reference(s) in Google Scholar.)
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