Rhodamine 123 is a fluorescent, monovalent cationic dye that serves as an indicator of mitochondrial membrane potential (ΔΨ). Its target is the mitochondrial inner membrane, across which it distributes in response to the membrane potential. It does not have a specific protein target with an associated IC50 or Ki value in this context [2].

The rhodamine 123 working solution is prepared. 1.1 Making the stock solution Take 1 milligram of rhodamine 123 and dissolve it in 525 μL DMSO to make a 5 mM stock solution. 1.2 Making the working solution for rhodamine 123 To create a working solution ranging from 1 to 20 μM, utilize serum-free cell culture medium or PBS stock solution. Note: The concentration of the Rhodamine 123 working solution should be adjusted based on the specific circumstances. 2. The staining of cells In a 6-well plate, 2.1 suspended cells a. Discard the supernatant after centrifuging at 1000 g for three to five minutes at 4°C. c. After adding 1 mL of the working solution, observe for one to three hours. c. Centrifuge at 400 g for three to four minutes at 4 °C; remove supernatant. d. Wash twice, for five minutes each time. One x 10^6 cells per milliliter. Wash for five minutes each time, twice, using PBS. e. Re-suspend cells in PBS or serum-free culture medium. either flow cytometry monitoring or fluorescence microscopy. 2.2 Adherent cells Adherent cells should be cultured on sterile glass slides (a). a. Take off the coverslip from the culture medium and use an aspirator to remove any extra. c. Add 100 μL working solution, give the cells a gentle shake to cover them fully, and leave them in place for 30 to 60 minutes. d. Use medium and wash twice in about five minutes. either flow cytometry monitoring or fluorescence microscopy. Note: Prior to staining, the cells must be resuspended if flow cytometry is being employed for detection.

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Self-Quenching Property: In aqueous solution, the fluorescence intensity of Rhodamine 123 is not linearly proportional to its concentration. It increases with concentration up to a peak, after which it decreases due to self-quenching. The peak intensity occurs at a concentration of approximately 50 μM when corrected for the inner filter effect (zero path length). This self-quenching behavior is critical for its use in mitochondria, as the dye concentrated in the matrix becomes quenched [2].
Mitochondrial Uptake and Fluorescence: In suspensions of isolated, energized mitochondria, Rhodamine 123 is taken up into the matrix and also partitions into the mitochondrial membrane. This uptake and binding lead to a decrease in overall fluorescence signal due to the high local concentration and consequent self-quenching of the dye in the matrix and potential quenching of membrane-bound dye. When the mitochondria are depolarized (e.g., by ADP addition or uncoupler CCCP), the dye is released back into the buffer, resulting in an increase in fluorescence intensity [2].

Mitochondrial Membrane Potential Assay in Isolated Mitochondria: The primary use of Rhodamine 123 detailed in this paper is to monitor changes in mitochondrial membrane potential (ΔΨ) in suspensions of isolated mitochondria. Guinea pig cardiac mitochondria (0.5 mg protein/mL) are suspended in a respiration buffer containing 10 mM pyruvate. R123 is added to a final concentration of 0.05 μM. The sample is placed in a spectrophotometer with excitation at 503 nm and emission at 527 nm, with continuous stirring. The decrease in fluorescence upon mitochondria addition indicates dye uptake. The addition of ADP (to initiate state 3 respiration) causes a transient mitochondrial depolarization, which is observed as a transient increase in fluorescence. Finally, the addition of the uncoupler CCCP (4 μM) completely collapses ΔΨ, causing a maximal increase in fluorescence, serving as a positive control for ΔΨ = 0. A model-based analysis of the fluorescence trace is required to estimate the actual ΔΨ transient [2].

Isolation of Cardiac Mitochondria from Guinea Pigs:** Guinea pigs (250-300 g) are anesthetized with ketamine. Ventricles are excised and rinsed in ice-cold isolation buffer (200 mM mannitol, 50 mM sucrose, 5 mM KH₂PO₄, 5 mM MOPS, 0.1% fatty acid-free BSA, 1 mM EGTA, pH 7.15). The tissue is minced and homogenized. The homogenate undergoes differential centrifugation: first at 8000×g for 10 min, the pellet is rinsed and re-centrifuged at 750×g for 10 min, and then the supernatant is collected and centrifuged at 8000×g. The final mitochondrial pellet is resuspended in isolation buffer (~10 mg protein/mL) and kept on ice. All procedures are carried out at 0-4°C. Mitochondrial protein is quantified using a protein assay with BSA as a standard [2].

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Isolation of Cardiac Mitochondria from Guinea Pigs: Guinea pigs (250-300 g) are anesthetized with ketamine. Ventricles are excised and rinsed in ice-cold isolation buffer (200 mM mannitol, 50 mM sucrose, 5 mM KH₂PO₄, 5 mM MOPS, 0.1% fatty acid-free BSA, 1 mM EGTA, pH 7.15). The tissue is minced and homogenized. The homogenate undergoes differential centrifugation: first at 8000×g for 10 min, the pellet is rinsed and re-centrifuged at 750×g for 10 min, and then the supernatant is collected and centrifuged at 8000×g. The final mitochondrial pellet is resuspended in isolation buffer (~10 mg protein/mL) and kept on ice. All procedures are carried out at 0-4°C. Mitochondrial protein is quantified using a protein assay with BSA as a standard [2].

Transport Kinetics: The distribution of Rhodamine 123 across the mitochondrial inner membrane is governed by its passive flux, modeled by the Goldman-Hodgkin-Katz equation. Its permeability coefficient (ρ) in guinea pig heart mitochondria is estimated to be 6.88 ± 0.39 mol (liter mitochondrial volume)⁻¹ s⁻¹ M⁻¹ [2].
Membrane Partitioning: Rhodamine 123 partitions into the mitochondrial membrane, described by a dimensionless partition coefficient (α). The estimated value of α is 4.49 ± 0.21 for guinea pig heart mitochondria [2].
Response Time: The fluorescence signal from R123 responds to rapid changes in mitochondrial membrane potential with a time constant of approximately 6 seconds, which is significantly slower than the actual change in ΔΨ (which occurs with a time constant of < 0.1 second upon ADP addition). This is because the signal is an aggregate of contributions from dye in the buffer, matrix, and membrane, and is limited by the transport kinetics [2].

Rhodamine 123 is noted for its low interference with underlying metabolic processes at the concentrations used for probing. However, a low total dye concentration (0.05 μM in this study) is used to minimize the impact of the R123 current itself on the inner membrane electrophysiology [2].

[1]. Emaus, R. K., Grunwald, R., & Lemasters, J. J. (1986). Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 850(3), 436–448.

[2]. Mitochondrial Inner Membrane Electrophysiology Assessed by Rhodamine-123 Transport and Fluorescence. Ann Biomed Eng. 2007 Jul; 35(7): 1276–1285.

A low-toxicity fluorescent probe is an effective substrate for ATP-binding cassette transporter B subfamily member 1 and bacterial multidrug efflux transporters. It can be used to assess mitochondrial bioenergetics in living cells and to measure the efflux activity of ATP-binding cassette transporter B subfamily member 1 in normal and malignant cells. (Leukemia 1997;11(7):1124-30)

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Rhodamine 123 is a widely used fluorescent dye for monitoring mitochondrial membrane potential (ΔΨ) due to its high sensitivity (high quantum yield), specificity, and non-invasive nature. It accumulates in the mitochondrial matrix of energized cells in response to the negative inside potential. Its utility relies on its property of self-quenching at high concentrations, which allows changes in its distribution (and thus fluorescence) to be correlated with changes in ΔΨ. Importantly, the relationship between measured fluorescence and ΔΨ is not linear but sigmoidal, and is highly sensitive to experimental conditions like dye and mitochondrial concentrations. A quantitative model is often necessary to accurately deconvolve ΔΨ transients from raw fluorescence data, as the fluorescence response is an order of magnitude slower than the actual bioenergetic events [2].

380.092

62669-70-9

9929799

Pink to red solid powder

235 °C

5.535

3

5

3

27

706

0

O1C2=C([H])/C(/C([H])=C([H])C2=C(C2=C([H])C([H])=C([H])C([H])=C2C(=O)OC([H])([H])[H])C2C([H])=C([H])C(=C([H])C1=2)N([H])[H])=N/[H]

TUFFYSFVSYUHPA-UHFFFAOYSA-M

InChI=1S/C21H17N2O3.ClH/c1-25-21(24)15-5-3-2-4-14(15)20-16-8-6-12(22)10-18(16)26-19-11-13(23)7-9-17(19)20/h2-11H,22-23H2,1H31H/q+1/p-1

Xanthylium, 3,6-diamino-9-(2-(methoxycarbonyl)phenyl)-, chloride

Rhodamine 123 R 22420 R 302 RH 123.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~62.5 mg/mL (~164.12 mM)

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.

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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).

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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)

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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).

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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

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)