MitoPedia: Sample preparations

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MitoPedia: Sample preparations

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

See also

O2k-Protocols: O2k-Mitochondrial preparations

Blood cell preparationbcpBlood cell preparation (bcp) is one of the key steps in diagnostic protocols.
Blood plasmaPlasmaBlood plasma is the non-cellular component of the blood. Plasma lacks cellular components of the blood, red blood cells, white blood cells, and platelets. However, there are many proteins in plasma, i.e. fibrinogen, albumin and globulin. Both blood plasma and platelet-rich plasma maintain clotting activity after whole blood separation.
Blood serumSerumBlood serum is a purified plasma in which the coagulant components were removed from the blood plasma. It contains other substances, i.e. antibodies, antigens and hormones. Serum can be obtained by collecting the liquid phase after blood or plasma coagulation.
Isolated mitochondriaimtIsolated mitochondria, imt, are mitochondria separated from a tissue or cells by breaking the plasma membranes and attachments to the cytoskeleton, followed by centrifugation steps to separate the mitochondria from other components.
Living cellsceCell viability in living cells should be >95 % for various experimental investigations, including cell respirometry. Viable cells (vce) are characterized by an intact plasma membrane barrier function. The total cell count (Nce) is the sum of viable cells (Nvce) and dead cells (Ndce). In contrast, the plasma membrane can be permeabilized selectively by mild detergents (digitonin), to obtain the mt-preparation of permeabilized cells used for cell ergometry. Living cells are frequently labelled as intact cells in the sense of the total cell count, but intact may suggest dual meanings of viable or unaffected by a disease or mitochondrial injury.
Mitochondrial concentrationCmtEMitochondrial concentration is CmtE = mtEV-1 [mtEU路m-3]. mt-Concentration is an experimental variable, dependent on sample concentration.
Mitochondrial contentmtENXMitochondrial content per object X is mtENX = mtENX-1 [mtEU路x-1].
Mitochondrial densityDmtESpecific mitochondrial density is DmtE = mtEmX-1 [mtEU路kg-1]. If the amount of mitochondria, mtE, is expressed as mitochondrial mass, then DmtE is the mass fraction of mitochondria in the sample. If mtE is expressed as mitochondrial volume, Vmt, and the mass of sample, mX, is replaced by volume of sample, VX, then DmtE is the volume fraction of mitochondria in the sample.
Mitochondrial preparationsmtprepMitochondrial preparations (mtprep) are isolated mitochondria (imt), tissue homogenate (thom), mechanically or chemically permeabilized tissue (permeabilized fibers, pfi) or permeabilized cells (pce). In mtprep the plasma membranes are either removed (imt) or mechanically (thom) and chemically permeabilized (pfi), while mitochondrial functional integrity and to a large extent mt-structure are maintained in incubation media optimized to support mitochondrial physiological performance. According to this definition, submitochondrial particles (smtp) are not a mtprep, since mitochondrial structure is altered although specific mitochondrial functions are preserved.
Permeabilized cellspcePermeabilized cells (pce) are mitochondrial preparations obtained by selectively permeabilizing the plasma membrane (e.g., with digitonin), for the exchange of soluble molecules between the cytosolic phase and external medium, without damaging the mt-membranes.

Permeabilized cells (pce) are, therefore, not any longer viable or living cells (ce), since the intactness of cells implies the intactness of the plasma membrane. Any typical quantiative cell viability test (trypan blue etc) evaluating the intactness of the plasma membrane, yields a 100% negative result on fully permeabilized cells.

For permeabilizing the cell plasma membranes chemically with digitonin, without damaging the mt-membranes, the optimum concentration of digitonin must be previously determinated. The protocol SUIT-010 is designed for the evaluation of optimum digitonin concentration for permeabilizing cells, a requirement to account for differences between cell types, the concentration of cells, and variability between batches of the natural product digitonin.
Permeabilized muscle fiberspfiPermeabilized muscle fibers (pfi) are used as a mitochondrial preparation in respirometry to access mitochondrial function comparable to isolated mitochondria (imt). pfi are obtained by selectively permeabilizing the plasma membrane mechanically and chemically (saponin), for the exchange of soluble molecules between the cytosolic phase and external medium, without damaging the mt-membranes.
禄 MitoPedia topic: Mitochondrial preparations
Permeabilized tissueptiPermeabilized tissue (pti, see also permeabilized muscle fibers, pfi) are mitochondrial preparations obtained by selectively permeabilizing the plasma membrane mechanically or chemically (e.g., with saponin), for the exchange of soluble molecules between the cytosolic phase and external medium, without damaging the mt-membranes.
Platelet-rich plasmaPRPPlatelet-rich plasma (PRP) is obtained as the upper layer at low-speed centrifugation (around 150-200 g), when white and red blood cells sediment and thus get separated from plasma containing the platelets. For further details see blood cell preparation.
SamplesA sample is one or more parts taken from an ensemble that is studied. A sample is either stored for later quantification or prepared and possibly separated into subsamples, which are enclosed in a system for qualitative or quantitative investigation. A pure sample S is a pure gas, pure liquid or pure solid of a defined elementary entity-type. A pure biological sample is a cell type, tissue, or organism without its solid, liquid or gaseous environment. Then the system used to investigate sample S contains only entities of entity-type S, and the volume VS [L] and mass mS [kg] of the pure (sub)sample S are identical to the volume V and mass m of the experimental system. A pure sample S may be mixed with other components to be investigated as a solution, mixture, or suspension, indicated by the symbol s in contrast to the pure sample S. A sample s is obtained in combination with other components, such that the volume Vs [L] and mass ms [kg] of the sample s are larger than the volume VS and mass mS of the pure sample S. For example, the number of cells Nce [Mx] can be counted in a sample s of a cell suspension, whereas the mass mce [mg] of cells requires a pure sample S of cells to be measured on a mass-balance. Clarity of statistical representation is improved, if the symbol N is used for the number of primary samples taken from a study group, and the symbol n is used for the number of subsamples studied as technical repeats.
Sample mass concentrationCmsSample mass concentration is Cms = msV-1 [kg路m-3].
Submitochondrial particlessmtpSubmitochondrial particles (smtp) consist of membrane fragments which retain most of the enzymatic machinery required in electron transfer and oxidative phosphorylation. Such membrane fragments are continuous closed vesicles formed by resealing of mt-membrane fragments after disruption of the mitochondrial structure. smtp are used to isolate the inner-membrane-bound ET pathway (mETS) from the upstream modules of the Electron transfer pathway (ETS) which are located in the mt-matrix and outer mt-membrane (transporters). smtp are obtained by treatment of mitochondria with membrane-dispersing agents such as digitonin at high concentration or by sonic irradiation.
SubsamplenSubsamples can be obtained (1) from a homogenous sample (e.g. cell suspension, tissue homogenate, isolated mitochondria), (2) as subsamples obtained by splitting a sample into comparable parts (e.g. permeabilized muscle fibres from a biopsy split into different chambers for repeated measurements), or (3) repetitive sampling (e.g. taking multiple biopsies) at a single time point. Subsamples may be used for (i) application of different types of assay (e.g. for measurement of respiration and enzyme activities), and (ii) a number of repetitions, n, of the same assay on the same sample.
Tissue homogenatethomA tissue homogenate (thom) is obtained through mechanical micro-disruption of fresh tissue and the cell membranes are mechanically permeabilized.
Wet massMwThe wet mass of a tissue or biological sample, obtained after blotting the sample to remove an arbitrary amount of water adhering externally to the sample.

O2k-Publications in the MiPMap

Isolated mitochondria
Permeabilized cells
Permeabilized tissue
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