- Currently Answered Questions from the »Oroboros Ecosystem Agenda 2020 Archive«
- Nov-19 Th
What is the optimal ADP concentration to evaluate OXPHOS capacity in permeabilized muscle fibers (pfi)?
For the evaluation of OXPHOS capacity, the limitation of respiratory fluxes must be avoided by using a kinetically-saturating ADP concentration. In permeabilized muscle fibers, an ADP concentration of 5 mM or higher is needed to reach kinetically-saturating levels.
- »Optimum ADP concentration in pfi«
- » Komlodi Timea« and » Doerrier Carolina«
- Nov-02 Mo
How can you measure mitochondrial membrane potential with O2k-Fluorometry?
The extrinsic fluorophores safranin, TMRM or Rhodamine 123 can be used to measure mt-membrane potential in mitochondrial preparations.
- »Safranin«, »TMRM«, »Rhodamine 123«
- communicated by »Komlodi Timea«, »Iglesias-Gonzalez Javier« and »Cecatto Cristiane«
- Oct-28 We
Flow or flux - what is the difference?
Rate can be expressed both as flow (I) and flux (J). Flow is used if the sample X can be quantified as count (NX; the number of cells, organisms), and changes proportionally with the size of X. Flux is a size-independent quantity where the rate is expressed per mass or volume of sample.
- »BEC2020.1: Mitochondrial physiology« - Box 2. Metabolic flows and fluxes: vectoral, vectorial, and scalar
- communicated by »Cecatto Cristiane« and »Komlodi Timea« and »Gnaiger Erich«
- Oct-22 Th
LEAK respiration relates to intrinsic uncoupling.
Non-phosphorylating LEAK respiration is measured in the absence of externally added uncouplers (i) in the absence of adenylates, (ii) after depletion of ADP, or (iii) after inhibition of ATP synthase or adenine nucleotide translocase.
- »BEC2020.1: Mitochondrial physiology« - Table 2. Terms on respiratory coupling and uncoupling
- communicated by »Komlodi Timea« and »Cecatto Cristiane« and »Gnaiger Erich«
- Oct-21 We
Human PBMC and Platelets - samples for high-resolution respirometry obtained by minimally invasive procedures.
- Oct-16 Fr
Explore mitochondrial pathways in PBMC and platelets
The following SUIT protocols are specifically optimized for PBMC and platelets
- »SUIT-001 O2 ce-pce D004« and »SUIT-002 O2 ce-pce D007a«
- communicated by »Cecatto Cristiane« and »Garcia-Souza Luiz F«
- Oct-15 Th
How can you reduce the amount of biological sample needed for high-resolution respirometry?
The O2k-sV-Module provides all the components necessary for a smaller operation volume (0.5 mL) whereby the sample amount used for measuring oxygen flux can be reduced.
- communicated by »Komlodi Timea« and »Schmitt Sabine«
- Oct-07 We
Background H2O2 calibration in the Amplex® UltraRed (AmR) assay:
A background H2O2 calibration is performed before addition of sample. This is required for (1) calculation of the chemical background fluorescence in our Excel analysis template, and (2) calculation AmR assay sensitivity, for quality control.
- »Background H2O2 calibration«
- communicated by »Komlodi Timea« and »Schmitt Sabine«
- Sep-16 We
Are permeabilized muscle fibers (pfi) a reliable model for studying ROS production?
High oxygen concentrations are needed to avoid oxygen limitation in pfi. However, high oxygen pressures may artificially increase ROS prodcution, including H2O2, making pfi a questionable model for ROS production.
- »Oxygen dependence of ROS production in pfi«
- communicated by »Komlodi Timea« and »Cardoso Luiza«
- Sep-10 Th
What is the limitation of State 3 respiration compared to OXPHOS capacity?
OXPHOS capacity is defined as the maximal respiratory capacity in the OXPHOS state in the presence of kinetically-saturating concentration of ADP, Pi, fuel substrates and O2. However, State 3 as defined by Chance and Williams (1955) can lead to underestimation of OXPHOS capacity and overestimation of excess E-P capacity due to kinetic limitations of the phosphorylation system.
- »BEC: Mitochondrial physiology« - Table 3 and several figures.
- communicated by » Cecatto Cristiane « and » Komlodi Timea «
- Aug-26 We
Non-mitochondrial respiration or residual oxygen consumption (Rox)? — Concept-driven constructive terminology frames our perception.
After inhibition of electron-transfer (ET) pathways or in the absence of fuel substrates, both mitochondrial non-ET reactions and non-mitochondrial reactions contribute to residual oxygen consumption in mt-preparations and living cells.
- »BEC: Mitochondrial physiology« — Table 1: Rox induced by non-ET pathway oxidation reactions
- Aug-24 Mo
Can NADH be used as an externally added substrate to obtain the NADH electron transfer-pathway state in mitochondrial respiration?
No, in most types of intact mitochondria NADH cannot pass the mitochondrial inner membrane (mtIM). Yeast mitochondria are an exception.
- »Nicotinamide adenine dinucleotide« and »Substrates and metabolites«
- Aug-20 Th
Would you like to simultaneously monitor O2 flux and the redox state of the Q-junction in isolated mitochondria? Soon you will be able to with the new Q-Module being developed as a part of the H2020 SME NextGen-O2k project:
- Aug-13 Th
Photosynthesis efficiency is reduced by 25% in plants and algae due the metabolic process called photorespiration.
- Aug-06 Th
OXPHOS-coupling efficiency (1-L/P) describes the coupling of the mitochondrial electron transfer system. This coupling control factor ranges from 1.0 for a fully coupled system to 0.0 for a system with zero respiratory phosphorylation capacity (L=P). It is suggested to use OXPHOS-coupling efficiency instead of the respiratory acceptor control ratio (RCR) to evaluate mitochondrial coupling.
- Jul-30 Th
Chlororespiration is the phenomenon by which oxygen is consumed in the chloroplasts of plant cells:
- Jul-24 Fr
Do you know how to assess the integrity of the mitochondrial outer membrane in mitochondrial preparations?
- Jul-16 Th
Do you know how to select the proper fluorescence sensors and filter set?
Using different fluorescence sensors, LED and photodiode filters, the O2k-Fluo Smart-Module (or O2k-Fluo LED2-Module) allows for measurement of hydrogen peroxide, ATP, mitochondrial membrane potential, calcium, and numerous other applications open for O2k-user innovation.
- »Selecting the Smart Fluo-Sensor« and »Selecting a Filter Set«
- Jul-09 Th
Do you know the different ways to adjust oxygen concentration in the Oroboros O2k?
- Jul-02 Th
Did you know that if ET capacity is lower than OXPHOS capacity (P>E) in living cells or mitochondrial preparations, then you have encountered an experimental artefact? The possible triggers of this experimental artefact are:
- loss of oxidative capacity during the time course of the respirometric assay,
- insufficient uncoupler concentrations,
- over titration of an uncoupler, which inhibits ET capacity,
- inhibition of ET capacity due to the use of high oligomycin (inhibitor of the ATP synthase) concentrations employed to measure LEAK respiration prior to ET capacity. Information from: Gnaiger Erich et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1 »Bioblast link«
- Jun-25 Th
Did you know that dithionite should not be used to control the oxygen concentration in experiments with biological samples?
- » Dithionite«
- Jun-18 Th
Did you know the Crabtree effect can affect respiration in living cells?
- Jun-11 Th
Did you know that some chemicals are incompatible with the Amplex UltraRed assay?
- Jun-08 Mo
Celebrating World Oceans Day
Would you like to measure the O2 flux that occurs during photosynthesis using the O2k and conduct experiments about primary productivity in aquatic ecosystems? Soon you will be able to with the PB-Module that we are developing as part of the H2020 NextGen-O2k project.
- Jun-04 Th
Mitochondria show variable preferences for glutamate & malate or pyruvate & malate to support NADH-linked respiration, depending on species, tissues, and pathophysiological state.
DatLab 7.4 provides a step-by-step guide through SUIT-008 and SUIT-014, which can be used in parallel to compare respiratory capacities supported by these NADH-linked fuel substrates.
-»SUIT-008« and »SUIT-014«
- May-28 Th
Did you know that oxygen concentration in the O2k-Chamber can be decreased by injecting nitrogen gas?
- May-21 Th
As part of the H2020 SME NextGen-O2k project we are developing a PhotoBiology module that will allow to study the effect of different light intensities on O2 consumption and production.
- May-14 Th
How can I measure ATP production with the O2k?
- May-07 Th
Do you know how to evaluate fatty acid oxidation (FAO, F-pathway) avoiding to overestimate it?
Did you know that malate alone might support mt-respiration in mitochondrial preparations, if malate anaplerotic pathways (e.g. mt-malic enzyme) are present?
- Learn more about - »malate-anaplerotic pathway control state«
- DatLab 7.4 provides a step-by-step guide through the SUIT-027 to evaluate the malate anaplerotic pathway - »SUIT-027«
- Since F-pathway can be overestimated due to anaplerotic reactions, SUIT-002 was specially designed to give information on F-pathway in OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways - »SUIT-002«
- Apr-24 Fr
ETC - the electron transport chain - is the most frequently applied misnomer in bioenergetics. Y Hatefi in the team of DE Green clarified this 58 years ago, introducing the concept of the electron transfer system.
- Apr-21 Tu
Citrate synthase activity is commonly applied as a mitochondrial marker - and erroneous applications are common in the literature.
- Apr-13 Mo
Is your mitochondrial preparation contaminated with other membranous organelles containing ATPases?
- Apr-08 We
The optimum effective digitonin concentration for complete plasma membrane permeabilization without mitochondrial outer membrane damage.
- Apr-02 Th
Do you want to search the best SUIT protocol for your research questions?
- Use the SUITbrowser: »SUITbrowser«
- Mar-16 Mo
The standardized coupling control protocol to explore the bioenergetics of living cells
DatLab 7.4 provides a step-by-step guide through the SUIT-003 protocol and its extensions.
- Mar-11 We
Does blebbistatin reduce oxygen dependence of respiration of permeabilized muscle fibers? This is one of the questions addressed in the COST Action CA15203 MitoEAGLE WG2.
- Mar-06 Fr
Explore metabolic profiles and specific modulations of mitochondrial pathways in health and disease with SUIT reference protocols.
- Mar-03 Tu
- Feb-26 We
It is essential to titrate uncouplers in multiple steps for respirometric measurement of ET capacity.
- Feb-24 Mo
More research is needed on the impact of viral infection on mitochondrial function.
- Feb-19 We
Spermidine and other calorie restriction mimetics control autophagy and mitochondrial function.
- Feb-18 Tu
Why do we fight gas bubbles?
- Dive deep into »Oxygen solubility«
- Feb-12 We
What do metformin, melatonin and other bioactive mitObesity compounds have in common?
- »MitObesity drugs«
- Jan-16 Th
- which SI units should be used?
Did you ever think that units for body mass could be an issue?
- »Body mass«
- Jan-08 We