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MitoPedia alert - 2024
- Currently Answered Questions from the »Oroboros Ecosystem Agenda 2023 Archive«
Dec 2024
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Metal chelation decreases the high background flux in the Amplex® UltraRed assay
DTPA (Diethylenetriaminepentaacetic acid) is a metal chelator that can be incorporated into the Amplex® UltraRed assay to decrease the high background fluxes associated with iron contamination.
- »DTPA«; »Amplex UltraRed«
- communicated by »Willis Jaime R« and »Grings Mateus«
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How can titration artifacts in the flux plot be hidden to have a more clear figure of the trace?
Select the corresponding plot in one of the graphs, press Ctrl+Shift+left click at the chosen starting time point and drag the cursor over the section to be hidden.
- »Hide/Show points - DatLab«
- communicated by »Grings Mateus« and »Baglivo Eleonora«
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Nov 2024
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MiR05 or MiR06?
MiR06 is MiR05 plus catalase. Including catalase in the medium allows for reoxygenation with H2O2 without needing to open the chamber, and it is recommended when measuring CIV activity (with ascorbate/TMPD).
- »Catalase«; »MiR05«; »MiR06«
- communicated by »Baglivo Eleonora« and »Leo Elettra«
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Living cells vs mitochondrial preparations
Information gained from simple respirometric protocols with living cells is limited compared to the analytical power of SUIT protocols with mitochondrial preparations, revealing the diversity of coupling and pathway control of mitochondrial respiration.
- »Electron-transfer-pathway state«
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Substrate-uncoupler-inhibitor titration (SUIT) reference protocols, RP1 and RP2, interrogate 20 mitochondrial pathway and coupling control states, including residual oxygen consumption for baseline correction, in different mitochondrial preparations.
- »SUIT-001«, »SUIT-002«
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Oct 2024
Sep 2024
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Proton pump or hydrogen ion pump?
Although used synonymously in chemistry, in particle physics a proton p+ is a positively charged subatomic particle and a hydrogen ion H+ is a positively charged ion (cation). CI, CIII, CIV, and ATP synthase are mitochondrial hydrogen ion pumps.
- »Hydrogen ion pump«; »Hydrogen ion«
- communicated by »Timon-Gomez Alba« and »Leo Elettra«
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There is no current cure for mitochondrial diseases
Most treatments and medication focus on alleviating the symptoms associated with the disease and slowing the course of the disease.
- »Mitochondrial disease«
- communicated by »Timon-Gomez Alba« and »Cardoso Luiza«
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What are coupling-control states?
They are defined as LEAK respiration, OXPHOS and ET states, with corresponding respiration rates (L, P, and E respectively), in the various mitochondrial preparations.
- »Coupling-control state«
- communicated by »Willis Jaime R« and »Baglivo Eleonora«
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How has the O2 calibration been improved in DatLab 8?
When R1 or R0 protocol marks are set, the calibration window pops up automatically. If a measurement with the same temperature and sensor number as the O2 calibration is performed within 24h, the box at the status bar will be green.
- » Oxygen calibration«, » Status bar«
- communicated by »Grings M« and »Baglivo E«
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Aug 2024
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Can I save my own graph layout in DatLab?
A modified standard layout can be saved as either a Lab or User layout. This is recommended when a team or an individual wants to adjust scalings, plots and graphs according to a specific sample or project.
- »Lab layouts«, »User layouts«
- communicated by »Baglivo Eleonora« and »Grings Mateus«
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Respiratory oxygen flow or flux?
Oxygen flow is the oxygen consumption per total system. An experimental object, e.g. a living cell, may be considered as the 'experimental system'. In this case, the respiratory oxygen flow is normalized for the cell count, differing from oxygen flux, which is oxygen flow normalized for system size, i.e. volume, mass, or a specific marker.
- »Oxygen flow«, »Oxygen flux«
- communicated by »Leo Elettra« and »Schmitt Sabine«
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Jul 2024
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Can my chemical solvent affect experimental results?
Some solvents (carriers) might alter mitochondrial physiology. To test for this effect, a control experiment with carrier titrations is needed in parallel to the treatment experiment.
- »Carrier control titrations«
- communicated by »Timon-Gomez Alba« and »Leo Elettra«
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Jun 2024
2024 Week 25
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How can I normalize data for the sample amount or concentration in DatLab 8?
In the 'Sample and medium' window, select the normalization type from a predefined list or set it manually, then enter the values for normalization.
- »Sample and medium - DatLab«
- communicated by »Grings Mateus« and »Baglivo Eleonora«
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Beyond O2 consumption — measuring ATP dynamics with Magnesium Green (MgG)
MgG fluoresces when bound to Mg2+. By taking advantage of the different binding affinities of ADP and ATP for Mg2+, we can follow ATP production across a variety of mitochondrial preparations. As ATP is produced, more Mg2+ is bound, and the MgG fluorescence decreases.
- »Magnesium Green« »Cardoso 2021 BEC MgG«
- communicated by »Willis Jaime R« and »Cardoso Luiza«
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May 2024
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How do I determine the optimal oligomycin concentration for my assay?
Perform the oligomycin titration test, and adjust your oligomycin concentration to minimize the inhibition of ET capacity.
- »Oligomycin titration test«
- communicated by »Baglivo Eleonora« and »Schmitt Sabine«
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What is the effective concentration of digitonin for complete plasma membrane permeabilization?
It needs to be determined for each cell type and digitonin batch. The SUIT protocol SUIT-010 was designed to evaluate the optimum digitonin concentration to permeabilize the plasma membrane.
- »Digitonin«, »SUIT-010«
- communicated by »Schmitt Sabine« and »Timon-Gomez Alba«
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Apr 2024
Mar 2024
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Why is it important to perform respirometry with permeabilized muscle fibers (pfi) under a high [O2] regime?
Long diffusion distances in the pfi result in an O2 diffusion limitation, which is overcome by increasing the O2 partial pressure inside the chamber. Proper instrumental background correction is a must and O2 dependence of ROS production should be considered.
- »Permeabilized muscle fibers«, »Oxygen flux - instrumental background«
- communicated by »Grings Mateus« and »Cardoso Luiza«
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Feb 2024
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What is the O2 solubility factor of the medium (FM) and why is it important?
FM expresses the effect of the salt concentration on oxygen solubility relative to pure water, and varies depending on the temperature and composition of the medium. The correct FM needs to be entered in DatLab when performing an air calibration.
- »Oxygen solubility factor« , »Air calibration in DatLab«
- communicated by »Baglivo Eleonora« and »Timon-Gomez Alba«
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Jan 2024
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Can the S-pathway O2 flux be derived by subtracting N-pathway O2 flux from NS-pathway O2 flux?
No, the sum of the O2 fluxes measured in the presence of NADH-linked substrates alone and succinate as the single substrate is typically higher than using a combination of all these substrates. This effect is described as incomplete additivity:
- »Additive effect of convergent electron flow«
- communicated by »Cardoso Luiza« and »Grings Mateus«
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New year, new state? What is REN state?
REN state is now distinguished from ROX state: REN is measured in mitochondrial preparations without addition of substrates, whereas ROX is due to oxidative side reactions remaining after inhibition of the electron transfer pathway.
- »REN«, »ROX«
- communicated by »Timon-Gomez Alba« and »Leo Elettra«
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Stay alert
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