A method for the counting of mitochondria (original) (raw)
Related papers
Analytical Biochemistry, 1978
The sedimentation profile (sediterm) of subcellular particles in homogenous media depends on the average sedimentation coefficient (s value) and the size distribution. The present study has focused on the two common types of polydispersity. i.e., (i) a variable standard deviation in a normal (Gaussian) size distribution, and (ii) two populations of particles with defined 3 values and size distributions. Theoretical considerations and experimental data indicate that rat liver mitochondria have a normal size distribution, (F = 0.391 2 0.063 pm) with much smaller standard deviation than previously assumed (U = 0. I 18 pm) based on isokinetic gradient centrifugation and electron microscopy. Sedimentation of a mixture of rat liver and guinea pig ileal mitochondria having the 3 values 17,040 S and 5640 S. respectively, gave the expected profile (sediterm) oftwo populations of particles. Their .!j values were estimated to be identical to those obtained when the individual mitochondrial populations were sedimented. The ratio between the populations (based on the assay of marker enzyme) was found to be identical to the expected value.
An Improved Method for Preparation of Uniform and Functional Mitochondria from Fresh Liver
Background and Aims: As the major energy source for mammalian cells, mitochondria have been the subject of numerous studies. However, the isolation and purification of healthy mitochondria, especially from fresh tissue, remains challenging. The most popular methods and kits involve various centrifugation steps which require substantial time and equipment but do not consistently provide pure preparations of functional mitochondria. The aim of this study was to determine whether methods could be devised to improve the purity and yield of functional mitochondria from fresh tissue. Methods: Fresh mouse liver was homogenized, and cells lysed. Particle size analysis, quantitation of mitochondrial DNA, mitochondrial oxygen consumption, and purity of mitochondria (by electron microscopy) were measured in samples after various purification steps and significant differences determined. Results: A two-step procedure consisting of centrifugation followed by filtration through 1.2m and 0.8m filters resulted in uniform mitochondrial preparations with diameters between 520-540 nm, and approximately 5-times more pure samples. The mitochondria thus obtained had oxygen consumption and sensitivities to mitochondrial inhibitors that were indistinguishable from those purified by centrifugation alone. Electron microscopy confirmed the presence of more uniform and 4-5 times greater concentrations of mitochondria compared to centrifugation alone. Conclusions: A two-step procedure consisting of sequential centrifugation followed by filtration is a rapid method for the production of highly purified, uniform and functional mitochondria.
Cancer Research, 1980
Mitochondria were isolated from whole homogenates of nor mal liver and Novikoff hepatomas using reorienting rate zonal centrifugation on sucrose gradients. The activities of several mitochondrial-specific enzymes and ultrastructure were com pared in the two tissues. Our results indicate that cytochrome oxidase, lipoamide dehydrogenase, malate dehydrogenase, and Buccinate dehydrogenase activities are all higher in liver homogenates than in Novikoff hepatoma homogenates. Mitochondrial hexokinase, however, is much greater in the hepa toma than in liver. The activity of these enzymes in isolated mitochondria displayed a much different pattern. Both cyto chrome oxidase and succinate dehydrogenase activities were higher in hepatoma mitochondria than in liver mitochondria. Lipoamide dehydrogenase and malate dehydrogenase, con versely, were higher in liver mitochondria. Hexokinase was found to be virtually absent in liver mitochondria but plentiful in hepatoma mitochondria. Ultrastructural studies have shown that the hepatoma mitochondria are much smaller in size, which results in a decreased rate of migration into the gradient. These studies have also shown that normal liver consists of predom inantly "condensed" forms of mitochondria, whereas hepa toma contained a majority of "twisted" species. Experiments using 1% bovine serum albumin in the homogenization proce dures and in the gradient have confirmed earlier observations that bovine serum albumin is essential for optimal isolation of neoplastic mitochondria.
The Journal of Cell Biology
The buoyant densities of the nuclear and mitochondrial DNA from the thoracic muscles of Sch~stocerca gregarm ,,-ere found to be 1 702 and 1.689 g/cm s, respectively, corresponding to guanine plus cytosine (G + C) content of 42.2 and 30% A prehminary treatment of the mitochondrial pellet with DNase (25°C, 20 min) is necessary to eliminate the contaminating nuclear DNA. The mitochondrml DNA renatures readily after heat denaturation and incubation at 65°C. The DNA released from the mltochondrial pellet by osmotic shock consists of circular open and closed molecules with a contour length around 5/~ The instability of insect mitochondrla in in vitro preparations is discussed.
Methods to detect mitochondrial function
Experimental Gerontology, 2004
The bioenergetic function of mitochondria can be investigated in intact cells by a variety of methods. A simple biochemical method to compare mitochondrial oxidative phosphorylation with glycolytic ATP synthesis takes advantage of the Pasteur effect, since the amount of lactate produced under basal conditions is an indication of glycolytic ATP, while the D-lactate (the difference between excess lactate produced after inhibition of respiration and basal lactate) represents ATP produced anaerobically in order to compensate for decreased oxidative phosphorylation after respiratory chain inhibition. The system has been validated in a series of cells, including human platelets and lymphocytes and lines cultured in vitro. Measurement of KCN-sensitive oxygen consumption by the cells and its sensitivity to uncouplers can be good supplementary indication of mitochondrial phosphorylative capacity.
Analysis of Mitochondria Isolated From Single Cells
Analytical and …, 2007
Bulk studies are not suitable to describe and study cell-to-cell variation, which is of high importance in biological processes such as embryogenesis, tissue differentiation, and disease. Previously, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to measure the properties of organelles isolated from millions of cells. As such, these bulk measurements reported average properties for the organelles of cell populations. Similar measurements for organelles released from single cells would be highly relevant to describe the subcellular variations among cells. Toward this goal, here we introduce an approach to analyze the mitochondria released from single mammalian cells. Osteosarcoma 143B cells are labeled with either the fluorescent mitochondrionspecific 10-N-nonyl acridine orange (NAO) or via expression of the fluorescent protein DsRed2. Subsequently, a single cell is introduced into the CE-LIF capillary where the organelles are released by a combined treatment of digitonin and trypsin. After this treatment, an electric field is applied and the released organelles electromigrate toward the LIF detector. From an electropherogram, the number of detected events per cell, their individual electrophoretic mobilities, and their individual fluorescence intensities are calculated. The results obtained from DsRed2 labeling, which is retained in intact mitochondria, and NAO labeling, which labels all mitochondria, are the basis for discussion of the strengths and limitations of this single-cell approach.
Staining of cellular mitochondria with LDS-751
Journal of Immunological Methods, 2001
We have found the dye LDS-751 to bind almost exclusively to mitochondria when incubated with viable, nucleated cells. Treatment of cells with the nuclear stain acridine orange and LDS-751 revealed little colocalization when the cells were examined by confocal microscopy. Staining with the dye rhodamine 123, which is known to bind polarized mitochondria, was virtually identical to the pattern observed with LDS-751. This staining pattern was observed to be consistent over a range of 0.02-20 mgrml LDS-751 and was consistent between both fibroblasts and monocytes. Depolarization of mitochondria with the mitochondrial depolarizing agents phenyl arsine oxide and carbonyl cyanide m-chlorophenylhy-Ž . drazone CCCP dramatically reduced both LDS-751 staining, and rhodamine 123 fluorescence. Taken together, these results suggest that LDS-751 is excluded from the nucleus and binds the polarized membranes of mitochondria. Given this, interpretation of LDS-751 fluorescence as being indicative of nuclear status, as is commonly done to discriminate between leukocytes and erythrocytes, is unwarranted and may lead to erroneous conclusions if mitochondria become depolarized upon processing. q