MitoPedia: Media for respirometry

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high-resolution terminology - matching measurements at high-resolution

MitoPedia: Media for respirometry

The MitoPedia terminology is developed continuously in the spirit of Gentle Science.

Mitochondrial respiration media are designed to support and preserve optimum mitochondrial function. The most important variables to be considered are: osmotic pressure, ionic strength, ion composition, Ca2+ and free fatty acid binding, and antioxidant capacity.

Air calibrationR1Air calibration of an oxygen sensor (polarographic oxygen sensor) is performed routinely on any day before starting a respirometric experiment. The volume fraction of oxygen in dry air is constant. An aqueous solution in equilibrium with air has the same partial pressure as that in water vapour saturated air. The water vapour is a function of temperature only. The partial oxygen pressure in aqueous solution in equilibrium with air is, therefore, a function of total barometric pressure and temperature. Bubbling an aqueous solution with air generates deviations from barometric pressure within small gas bubbles and is, therefore, not recommended. To equilibrate an aqueous solution ata known partial pressure of oxygen [kPa], the aqueous solution is stirred rigorously in a chamber enclosing air at constant temperature. The concentration of oxygen, cO2 [µM], is obtained at any partial pressure by multiplying the partial pressure by the oxygen solubility, SO2 [µM/kPa]. SO2 is a function of temperature and composition of the salt solution, and is thus a function of the experimental medium. The solubility factor of the medium, FM, expresses the oxygen solubility relative to pure water at any experimental temperature. FM is 0.89 in serum (37 °C) and 0.92 in MiR06 or MiR05 (30 °C and 37 °C).
Biopsy preservation solutionBIOPSBiopsy preservation solution, for preservation of tissue samples, preparation of muscle fibres, and permeabilization with saponin.
Buffer ZBuffer ZMitochondrial respiration medium, Buffer Z, described by Perry 2011 Biochem J For composition and comparison see: Mitochondrial respiration media: comparison
Cell culture mediaCell culture media, like RPMI or DMEM, used for HRR of living cells.
MiP03MiP03Mitochondrial Preservation Medium, MiP03, developed for preservation of isolated mitochondria.
MiR05MiR05Mitochondrial respiration medium, MiR05, developed for oxygraph incubations of mitochondrial preparations. Respiration of living cells may be assessed in MiR05 by adding pyruvate (P) as an external source. MiR06 = MiR05 + catalase. MiR05Cr = MiR05 + creatine.
MiR05CrMiR05CrMitochondrial respiration medium, MiR05Cr, developed for oxygraph incubations of mitochondrial preparations - permeabilized muscle fibers. MiR05Cr = MiR05 + 20 mM creatine.
MiR06MiR06Mitochondrial respiration medium, MiR06, developed for oxygraph incubations of mitochondrial preparations. MiR06 = MiR05 plus catalase. MiR06Cr = MiR06 plus creatine.
MiR06CrMiR06CrMitochondrial respiration medium, MiR06Cr, developed for oxygraph incubations of mitochondrial preparations - permeabilized muscle fibers. MiR06Cr = MiR06 + 20 mM creatine.
MiRK03MiRK03Mitochondrial respiration medium, MiRK03, modified after a medium described by Komary 2010 Biochim Biophys Acta, intended for use as medium for H2O2 production measurement with Amplex Red.
MitoOx1MitoOx1Mitochondrial respiration medium, MitoOx1, used by the Budapest groups for respirometry und Amplex Red trials.
MitoOx2MitoOx2Mitochondrial respiration medium, MitoOx2, developed for oxygraph incubations of mitochondrial preparations to measure the H2O2 production. MitoOx2 yields a higher optical sensitivity and lower "drift" (oxidation of the fluorophore precurcor without H2O2 present) for Amplex UltraRed(R) than e.g. MiR05.
Mitochondrial respiration media: comparisonMiRMitochondrial respiratory capacity and control are compared in different mitochondrial respiration media, MiRs, to evaluate the quality of MiRs in preserving mitochondrial function and to harmonize results obtained in various studies using different MiRs. In some cases alterations of the formulation are incorporated to optimize conditions for the simultaneous measurement of multiple parameters, e.g. respiration and ROS production.
Oxygen solubilitySO2 [µM/kPa]The oxygen solubility, SO2 [µM/kPa] = [(µmol·L-1)/kPa], expresses the oxygen concentration in solution in equilibrium with the oxygen pressure in a gas phase, as a function of temperature and composition of the solution. The inverse of oxygen solubility is related to the activity of dissolved oxygen. The oxygen solubility in solution, SO2(aq), depends on temperature and the concentrations of solutes in solution, whereas the dissolved oxygen concentration at equilibrium with air, cO2*(aq), depends on SO2(aq), barometric pressure and temperature. SO2(aq) in pure water is 10.56 µM/kPa at 37 °C and 12.56 µM/kPa at 25 °C. At standard barometric pressure (100 kPa), cO2*(aq) is 207.3 µM at 37 °C (19.6 kPa partial oxygen pressure) or 254.7 µM at 25 °C (20.3 kPa partial oxygen pressure). In MiR05 and serum, the corresponding saturation concentrations are lower due to the oxygen solubility factor: 191 and 184 µM at 37 °C or 234 and 227 µM at 25 °C.
Oxygen solubility factorFMThe oxygen solubility factor of the incubation medium, FM, expresses the effect of the salt concentration on oxygen solubility relative to pure water. In mitochondrial respiration medium MiR05, MiR05-Kit and MiR06, FM is 0.92 (determined at 30 and 37 °C) and in culture media is 0.89 (at 37 °C). FM varies depending on the temperature and composition of the medium. To determine the FM based on the oxygen concentration, specific methods and equipment are needed (see references Rasmussen HN, Rasmussen UF 2003 in MiPNet06.03). For other media, FM may be estimated using Table 4 in MiPNet06.03. For this purpose KCl based media can be described as "seawater" of varying salinity. The original data on sucrose and KCl-media (Reynafarje et al 1985), however, have been critizesed as artefacts and the FM of 0.92 is suggested in the temperature range of 10 °C to 40 °C as for MiR05.
PHpHThe pH value or pH is the negative of the base 10 logarithm of the activity of protons (hydrogen ions, H+). A pH electrode reports the pH and is sensitive to the activity of H+. In dilute solutions, the hydrogen ion activity is approximately equal to the hydrogen ion concentration. The symbol pH stems from the term potentia hydrogenii.
PH calibration bufferspH calibration buffers are prepared to obtain two or more defined pH values for calibration of pH electrodes and pH indicator dyes.
Sodium phosphate bufferNa-PBSodium phosphate buffer, Na-PB, for HRR with freeze-dried baker´s yeast.
Water, H2O, is widely used in the laboratory, particularly as a solvent and cleaning agent. Chemically pure water is prepared in various grades of purification: double distilled water (ddH2O) versus distilled water (dH2O or aqua destillata, a.d.) and deionized or demineralized water (diH2O) with various combination purification methods. When H2O is mentioned without further specification in published protocols, it is frequently assumed that the standards of each laboratory are applied as to the quality of purified water. Purification is not only to be controlled with respect to salt content and corresponding electrical conductivity (ultra-pure water: 5.5 μS/m due to H+ and OH- ions), but also in terms of microbial contamination.
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