Rhodamine 123

high-resolution terminology - matching measurements at high-resolution

Rhodamine 123

Description

Rhodamine 123 (Rh123) is an extrinsic fluorophore and can be used as a probe to determine changes in mitochondrial membrane potential. Rh123 is a lipophilic cation that is accumulated by mitochondria in proportion to Δψ_mt. Using ethanol as the solvent, the excitation maximum is 511 nm and the emission maximum is 534 nm.The recommended excitation and emission wavelengths in PBS are 488 and 515-575 nm, respectively (Sigma-Aldrich).

Abbreviation: Rh123

Application in HRR

Life Technologies, R302, 25 mg, store at -20 °C, FW=380.83 g/mol

Preparation of 5 mM storage solution (dissolved in ethanol)

1. Weight 1.9 mg of Rh123 and dissolve in 1 mL ethanol;
2. Divide into 0.1 mL aliquots(use dark vials);
3. Store at -20 °C.

Preparation of 0.2 mM stock solution

1. Dilute a 100 µL aliquot 5 mM storage solution with 2400 µL ethanol to reach 2.5 mL final volume;
2. Divide into 0.2 mL aliquots(use dark vials);
3. Store at -20 °C.

O2k manual titrations MiPNet09.12 O2k-Titrations

• Titration volume: in 2-µL steps using a 10 µL syringe (2 mL O2k-Chamber).
• Final concentration: 0.2-1 µM. Stepwise titration for calibration.

Signal and output

1. Signal: The O2k-Fluo LED2-Module is operated through the Amperometric (Amp)-Channel of the O2k, with electric current (ampere [A]) as the primary signal.
2. Fluo-Sensor: Smart Fluo-Sensor Blue equipped with Filter Set Saf
3. Output: The focus of the output with Rh123 is on Type III: Force, mt-membrane potential.

Rhodamine 123 chemical background

Several substances typically used in SUIT protocols may influence the fluorescence signal of safranin when injected into the O2k-Chamber (for instance colored substances such as cytochrome c). These chemicals should be tested for their effect in a background run without a biological sample, and the necessary corrections are applied.

Calibration of the raw fluorescence signal

To calibrate the raw fluorescence signal expressed as [V] and convert to µM, you need to click on ´Calibration/A: or B: Amperometric, Amp´ to open´[Amp calibration-DatLab| Amp calibration]´ window in DatLab.

-See:Krumschnabel 2014 Methods Enzymol

Rh123 with the O2k-FluoRespirometer: exploratory experiments

Figure 1. Simultaneous measurement of respiration and mt-membrane potential using Rhodamine 123. Brain mitochondria isolated from mouse were fueled with succinate (S-pathway control state) in presence of rotenone to inhibit Complex I, displaying an accumulation of the dye according to their Δψ_mt in the LEAK state (hyperpolarization, Amp record shows a decrease). After the addition of ADP, the depolarization of Δψ_mt is reflected in an increase of the fluorescence. The addition of the ATP synthase inhibitor oligomycin promotes to reach again LEAK state and, as consequence, hyperpolarization of the membrane (Amp record shows a decrease of the signal). Finally, upon the addition of the uncoupler CCCP, the Δψ_mt collapses and the release of the Rhodamine 123 from the mitochondria is associated with an increase of fluorescence. The fluorescence signal was calibrated by Rhodamine 123 before adding the sample in a step-wise titration (max. concentration 1 µM). The optimum concentration of Rhodamine 123 to measure the membrane potential has to be tested in previous experiments.

Bioblast links: Force and membrane potential - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

Fundamental relationships

» Force

» Affinity

» Flux

» Advancement

» Advancement per volume

» Stoichiometric number

mt-Membrane potential and protonmotive force

» Protonmotive force

» Mitochondrial membrane potential

» Chemical potential

» Faraday constant

» Format

» Uncoupler

O2k-Potentiometry

» O2k-Catalogue: O2k-TPP+ ISE-Module

» O2k-Manual: MiPNet15.03 O2k-MultiSensor-ISE

» TPP - O2k-Procedures: Tetraphenylphosphonium

» Specifications: MiPNet15.08 TPP electrode

» Poster

» Unspecific binding of TPP+

» TPP+ inhibitory effect

O2k-Fluorometry

» O2k-Catalogue: O2k-FluoRespirometer

» O2k-Manual: MiPNet22.11 O2k-FluoRespirometer manual

» Safranin - O2k-Procedures: MiPNet20.13 Safranin mt-membranepotential / Safranin

» TMRM - O2k-Procedures: TMRM

O2k-Publications

» O2k-Publications: mt-Membrane potential

» O2k-Publications: Coupling efficiency;uncoupling

MitoPedia methods: Fluorometry 

Labels: MiParea: Respiration, Instruments;methods 

HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Protocol 

O2k-MultiSensor