TMRM

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TMRM

Description

TMRM (tetramethylrhodamine methyl ester) is an extrinsic fluorophore and can be used as a probe to determine changes in mitochondrial membrane potential. TMRM is a lipophilic cation that is accumulated by mitochondria in proportion to Δψmt. Upon accumulation of the dye it exhibits a red shift in its absorption and fluorescence emission spectrum. The fluorescence intensity is quenched when the dye is accumulated by mitochondria.

Abbreviation: TMRM

Reference: Ehrenberg 1988 Biophys J


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Click to expand or collaps
» Keywords: Force and membrane potential
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


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O2k-technical support

This information is part of O2k-technical support and open innovation.

Application in HRR

TMRM: Tetramethyrhodamine methyl ester perchlorite (C25H25CIN2O7);
Sigma T 5428 or ThermoFischer T 668; 25 mg, store at -20°C; FW = 500.93
Caution: Light sensitive (store solution in a dark vial)!
Preparation of 5 mM storage solution (dissolved in DMSO)
  1. Weight 2.5 mg of TMRM and dissolve in 1 mL DMSO;
  2. Divide into 0.2 mL portions (use dark vials);
  3. Store at -20 °C.
Preparation of 1 mM stock solution
  1. Dilute a 200 µL aliquot 5 mM storage solution with 800 µL DMSO to reach 1 mL final volume;
  2. Divide into 0.2 mL portions (use dark vials);
  3. Store at -20 °C.


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. Output: The focus of the output with TMRM is on Type C: Force, mt-membrane potential.

TMRM with the O2k-FluoRespirometer: exploratory experiments

  • 100 mM potassium phosphate buffer, T=37°C
  • Fluorescence-Sensor Green, Filter Set AmR, light intensity: Amp Polarization voltage = 100, Amp Gain = 1000
  • TMRM perchlorate stock: 100 µM in DMSO
  • Titration TIP: 5x 0.2 µL (5x10 nM), 5 x 1 µL (5 x 50 nM), 7 x 2 µL (7 x 100 nM); interval: 120 s

TMRM 1mA Gain 1000.png

TMRM with the O2k-FluoRespirometer: experiments with mouse liver mitochondria by C. Chinopoulos

  • Mouse liver mitochondria
  • Protein (BCA assay) 130.5 mg/ml
  • O2k chamber: 0.5 mg/mL (0.489 mg/mL)
  • Experimental buffer composition: KCl 8 mM, K-gluconate 110 mM, Mannitol 10 mM, NaCl 10 mM, Hepes (free acid) 10 mM, K2HPO4 10 mM, K-EGTA 0.01 mM, BSA 0.5 mg/mL, pH 7.25 (KOH), MgCl2 1 mM (added in the buffer)
  • Glutamate: 10 mM (40 µL of a 1 M stock)
  • Malate: 2 mM (16 µL of a 0.5 M stock)
  • Pyruvate: 5 mM (20 µL of a 1 M stock)
  • Carboxyatractyloside (cATR): 0.5 µM (2 µL of a 1 mM stock)
  • SF6847: 0.5 µM (2 µL of a 1 mM stock)
  • ADP: 2 mM (40 µL of a 200 mM stock)

Experimental conditions:

  • 4 mL chamber volume
  • Fluorescence-Sensor Green, Filter Set AmR, light intensity: Amp Polarization voltage = 1000 (equivalent to level setting "6" on the Fluorescence module Series A front panel), gain 1000; T = 37.0 °C
  • TMRM was added 20-30 s before addition of mt to the O2k chamber.

Figure legends

Concurrent measurement of respiration and mitochondrial membrane potential (mtMP) in mouse liver mitochondria (MLmt): 0.5 mg/mL MLmt were added to the O2k chamber with 4 mL experimental buffer (composition see above) including pyruvate, glutamate and malate and containing TMRM at variable concentrations as indicated. After 100 s 2 mM ADP was added, followed by 0.5 mM CATR at 350 s and 0.5 µM SF6847 at 450 s. The concentration of the uncoupler SF6847 used was twice the concentration required to induce maximum oxygen flux so as to ensure maximum depolarization of mtMP.
Analysis of respiratory rates indicated that neither LEAK nor OXPHOS rates were significantly affected by TMRM up to 4 µM, with an RCR of at least 15 in each case. At 5 µM TMRM LEAK appeared to be elevated and RCR accordingly diminished to a value of 12.
Please note the very different scaling for the fluorescence signal in the individual graphs! The range is always described by the ∆=XY at the end of the legend.


TMRM Fig 1.png

Figure 1. Zero nM TMRM. Control measurement of MLmt in the absence of TMRM showing control respiratory flux (A; blue line: oxygen concentration, red line: oxygen flux) and background fluorescence (B; green line: raw signal of fluorescence, red line: rate of change in fluorescence). The maximum change in fluorescence induced by addition of chemicals amounts to approx. 0.003 fluorescence units (FU) (∆=0.003 FU).



TMRM Fig 2.png

Figure 2. 20 nM TMRM. MLmt incubated with 20 nM TMRM and energized by NADH-linked substrates display a basal level of fluorescence due to accumulation of the dye according to their mtMP in the LEAK state. Upon addition of ADP the proton motive force is partially dissipated to drive the generation of ATP (OXPHOS state) and this leads to a decrease of mtMP. A decrease of mtMP causes the release of accumulated TMRM and this is reflected by a decrease of the fluorescence signal. Addition of the inhibitor of the adenine nucleotide translocator (ANT) carboxyatractyloside (cATR) inhibits ATP synthesis (LEAK in the presence of ATP) and thus the influx of H+ through the ATP synthase (in addition to the fact that cATR stops the electrogenic exchange of ATP4- for ADP3-, thus sparing the loss of a negative charge per each exchange). The consecutive slow restoration of mtMP causes a re-accumulation of TMRM and hence an increase of the fluorescence signal. Finally, upon addition of the uncoupler SF6847 a collapse of mtMP is observed and thus a release of TMRM with an associated decrease of fluorescence. The short-lived peak of fluorescence after addition of uncoupler is due to transient unquenching of matrix TMRM caused by the rapid release of the dye. ∆=0.008 FU.


TMRM Fig 3.png

Figure 3. 50 nM TMRM. MLmt incubated with 50 nM TMRM, other conditions as in Fig. 2. ∆=0.016 FU.



TMRM Fig 4.png

Figure 4. 200 nM TMRM. MLmt incubated with 200 nM TMRM, other conditions as in Fig. 2. Short-lived peaks of fluorescence observed after addition of chemicals are due to transient unquenching of matrix TMRM caused by the rapid release of the dye. ∆=0.100 FU.



TMRM Fig 5.png

Figure 5. 500 nM TMRM. MLmt incubated with 500 nM TMRM, other conditions as in Fig. 2. At this concentration TMRM is at the verge of alternating between a response in quenching and unquenching mode. In the latter mode the relation between mtMP and TMRM concentration in the matrix becomes inverted in that a decrease of mtMP leads to an increase of fluorescence due to unquenching of fluorescence upon release of the dye. ∆=0.16 FU.


TMRM Fig 6.png

Figure 6. 1000 nM TMRM. MLmt incubated with 1000 nM TMRM, other conditions as in Fig. 2. In the unquenching mode the decrease of mtMP resulting from addition of ADP is mirrored by an increase of fluorescence, while the increase of mtMP due to inhibition of ATP synthesis is seen as a decrease of fluorescence. The collapse of mtMP is again associated with an increased fluorescence signal. ∆=0.295 FU.



TMRM Fig 7.png

Figure 7. 2000 nM TMRM. MLmt incubated with 2000 nM TMRM, other conditions as in Fig. 2, for explanation of changes in mtMP and fluorescence see legend to Fig. 6. ∆=0.956 FU.



TMRM Fig 8.png

Figure 8. 5000 nM TMRM. MLmt incubated with 5000 nM TMRM, other conditions as in Fig. 2, for explanation of changes in mtMP and fluorescence see legend to Fig. 6. The instability of the signal in the presence of ADP may reflect the onset of toxicity at this concentration of TMRM, possibly resulting in the opening of the permeability transition pore of a sub-population of mitochondria. ∆=2.601 FU.



TMRM Fig 9.png

Figure 9. 4000 nM TMRM. MLmt incubated with 4000 nM TMRM, other conditions as in Fig. 2, for explanation of changes in mtMP and fluorescence see legend to Fig. 6. ∆=2.535 FU.



TMRM Fig 10.png

Figure 10. 0 nM TMRM. Control measurement in the absence of TMRM. Conditions as in Fig. 1. ∆=0.070 FU.



TMRM Fig 11.png

Figure 11. 0 nM TMRM. Control measurement in the absence of TMRM. Conditions as in Fig. 1. ∆=0.015 FU.



TMRM with the O2k-FluoRespirometer: Experiments with rat liver homogenate by G. Krumschnabel and Z. Sumbalova

  • Wistar rat liver tissue homogenate
1 mg Ww/mlL
  • Experimental buffer: MiR05
  • Glutamate: 10 mM; Pyruvate: 5 mM; Malate: 2 mM; ADP: 2.5 mM; Succinate: 10 mM; CCCP (U) added in 0.5 µM steps; Rotenone: 1 µg/mL; Antimycin A: 2.5 µM; TMRM: 2 µM
Experimental conditions
2 mL chamber volume, Fluorescence-Sensor Green, Filter Set AmR, light intensity: Amp Polarization voltage = 500, gain 1000, T = 37.0 °C


TMRM Thom rat liver.png

Figure legends
Concurrent measurement of respiration [upper panel; pmol/(s*mL)] and mitochondrial membrane potential (mtMP) (lower panel; arbitrary units) in tissue homogenate (thom) from mouse liver: 1 mg/mL thom was added to the O2k chamber with 2 mL experimental buffer and then 2 µM TMRM were added as indicated. This was followed by addition of glutamate, pyruvate and malate to induce LEAK respiration. Next, 2.5 mM ADP was added to elicit NADH-linked OXPHOS, then succinate was injected to obtain NS-linked OXPHOS, before titrating uncoupler CCCP (U) in 0.5 µM steps to induce noncoupled respiration and collapse the mtMP. It should be noted that the CCCP concentration required for complete collapse of mtMP was higher than that needed for maximum respiration and that respiration was already inhibited at this higher CCCP. Finally, CI-inhibitor rotenone (Rot) and CIII-inhibitor antimycin A (Ama) were added to obtain residual oxygen consumption (Rox), which however did not further affect mtMP.


TMRM with the O2k-FluoRespirometer: Experiments with permeabilized HEK 293T cells by M. Hansl and G. Krumschnabel

  • HEK 293T cells
  • 1.5 106 cells/mL
  • Experimental buffer: MiR05
  • Pyruvate: 5 mM; Malate: 2 mM; ADP: 2.5 mM; Succinate: 10 mM; CCCP (U) added in 0.5-1 µM steps; Rotenone: 1 µg/mL; Antimycin A: 2.5 µM; TMRM: 2 µM final concentration
Experimental conditions
2 mL chamber volume, Fluorescence-Sensor Green, Filter Set AmR, light intensity: Amp Polarization voltage = 500, gain 1000, T = 37.0 °C


TMRM permHEK293T.png

Figure legends
Simultaneous measurement of respiration [upper panel; pmol/(s*mL)] and mitochondrial membrane potential (mtMP) (lower panel; arbitrary units) in permeabilized HEK 293T cells: TMRM was added to the O2k chamber with 2 mL experimental buffer in two 1 µL steps to obtain 2 µM TMRM and then 1.5 106 cells/mL were added. Next, cells were permeabilized by adding 10 µg/mL digitonin, followed by addition of pyruvate and malate to induce LEAK respiration. Then, 2.5 mM ADP was added to elicit N-linked OXPHOS, then succinate was injected to obtain NS-linked OXPHOS, before titrating uncoupler CCCP (U) in 0.5-1 µM steps to induce noncoupled respiration and collapse the mtMP. It should be noted that in the permeabilized cells CCCP concentration required for complete collapse of mtMP was lower than that needed for maximum respiration. Finally, CI-inhibitor rotenone (Rot) and CIII-inhibitor antimycin A (Ama) were added to obtain residual oxygen consumption (Rox), which however did not further affect mtMP.



TMRM with the O2k-FluoRespirometer: Experiments with isolated mouse cardiac muscle mitochondria by M. Spinazzi and G. Krumschnabel

  • Isolated mouse cardiac muscle mitochondria (provided by C. Doerrier)
  • Experimental buffer: MiR05
  • Pyruvate: 5 mM; Malate: 2 mM; ADP: 2.5 mM; Glutamate 10 mM; Succinate: 10 mM; CCCP (U) added in 0.5-1 µM steps; Rotenone: 1 µg/mL; Antimycin A: 2.5 µM; TMRM: 2 µM final concentration
Experimental conditions
2 mL chamber volume, Fluorescence-Sensor Green, Filter Set AmR, light intensity: Amp Polarization voltage = 200, gain 1000, T = 37.0 °C


TMRM Imt mouse heart.png

Figure legends
Respiration [upper panel; pmol/(s*mL)] and mitochondrial membrane potential (mtMP) (lower panel; arbitrary units) in isolated mouse cardiac mitochondria: TMRM was added to the O2k chamber with 2 mL experimental buffer in two 1 µL steps to obtain 2 µM TMRM and then mitochondria were added. This was followed by addition of pyruvate and malate to induce LEAK respiration and by addition of 2.5 mM ADP to elicit N-linked OXPHOS. Subsequently glutamate was injected inducing enhanced N-linked OXPHOS. Addition of succinate led to NS-linked OXPHOS, before titrating uncoupler CCCP (U) in 0.5-1 µM steps to induce noncoupled respiration and collapse the mtMP. As with rat liver homogenate (see above), the CCCP concentration required for complete collapse of mtMP superseded that needed for maximum respiration which was already inhibited at this higher CCCP. Finally, CI-inhibitor rotenone (Rot) and CIII-inhibitor antimycin A (Ama) were added to obtain residual oxygen consumption (ROX), which had no further affect on mtMP. Fluorescence signals were distorted during re-oxygenation and were thus deleted.


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Labels: MiParea: Respiration, Instruments;methods 





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