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| '''TMRM experiments by C. Chinopulos:'''
| | [[Image:BB-Bioblast.jpg|left|40px|link=Bioblast:About|Bioblast wiki]] |
| | | == Popular Bioblast page == |
| Mouse liver mitochondria
| | ::: [[TMRM]] has been accessed more than |
| | | ::::* 5,000 times (2019-12-16) |
| Protein (BCA assay) 130.5 mg/ml
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| O2k chamber: 0.5 mg/ml (0.489 mg/ml)
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| Experimental buffer composition:
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| 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)
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| Glutamate: 10 mM (40 µl of a 1 M stock)
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| Malate: 2 mM (16 µl of a 0.5 M stock)
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| Pyruvate: 5 mM (20 µl of a 1 M stock)
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| Carboxy atractyloside (CATR): 0.5 µM (2 µl of a (1 mM stock)
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| SF6847: 0.5 µM (2 µl of a 1 mM stock)
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| ADP: 2mM (40 µl of a 200 mM stock)
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| '''Experimental conditions:'''
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| 4ml chamber volume
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| [[Fluorescence-Sensor_Green|Fluorescence-Sensor Green]], [[Filter_Set_AmR|Filter Set AmR]], level setting "6" on the fluorimeter front panel, gain 1000
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| T = 37.0 °C
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| [[TMRM|TMRM]] was added 20-30 sec before addition of mitos to the Oxygraph chamber
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| '''Figure legends:'''
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| Concurrent measurement of respiration and mitochondrial membrane potential (mtMP) in mouse liver mitochondria (MLM): 0.5 mg/ml MLM 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.
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| Analysis of respiratory rates indicated that neither LEAK nor OXPHOS rates were significantly affected by TMRM up to 4000 nM, with an RCR of at least 15 in each case. At 5000 nM TMRM LEAK appeared to be elevated and RCR accordingly diminished to a value of 12.
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| [[Image:TMRM Fig 1.png|600px]]
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| '''Figure 1. Zero nM TMRM.''' Control measurement of MLM in the absence of TMRM showing control respiratory rates (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).
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| [[Image:TMRM Fig 2.png|600px]]
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| '''Figure2. 20 nM TMRM.''' MLM incubated with 20 nM TMRM and energized by CI-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 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) carboxy atractyloside (CATR) inhibits ATP synthesis (LEAK in the presence of ATP) and thus the influx of H+ through the ATP synthase. 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.
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| [[Image:TMRM Fig 3.png|600px]]
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| '''Figure 3. 50 nM TMRM.''' MLM incubated with 50 nM TMRM, other conditions as in Fig. 2. ∆=0.016 FU.
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| [[Image:TMRM Fig 4.png|600px]]
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| '''Figure 4. 200 nM TMRM.''' MLM 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.
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| [[Image:TMRM Fig 5.png|600px]]
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| '''Figure 5. 500 nM TMRM.''' MLM 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. ∆=0.016 FU.
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| [[Image:TMRM Fig 6.png|600px]]
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| '''Figure 6. 1000 nM TMRM.''' MLM 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.
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| [[Image:TMRM Fig 7.png|600px]] | |
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| '''Figure 7. 2000 nM TMRM.''' MLM 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.
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| [[Image:TMRM Fig 8.png|600px]]
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| '''Figure 8. 5000 nM TMRM.''' MLM 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.
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| [[Image:TMRM Fig 9.png|600px]]
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| '''Figure 9. 4000 nM TMRM.''' MLM 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.
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| [[Image:TMRM Fig 10.png|600px]]
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| '''Figure 10. 0 nM TMRM.''' Control measurement in the absence of TMRM. Conditions as in Fig. 1. ∆=0.070 FU.
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| [[Image:TMRM Fig 11.png|600px]]
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| '''Figure 11. 0 nM TMRM.''' Control measurement in the absence of TMRM. Conditions as in Fig. 1. ∆=0.015 FU.
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