In search of an effective cell disruption method to isolate intact mitochondria from Chinese hamster ovary cells

An efficient isolation of mitochondria from cells under physiological conditions is crucial for many studies in life sciences but still challenging in many cases such as in metabolic characterization of mitochondria. In this work, four methods for the disruption of Chinese hamster ovary cells were evaluated regarding their influence on mitochondrial integrity and yield. After cell disruption, mitochondria released from cells were separated from the remaining cell homogenate by differential centrifugation. Sonication was shown to be a rapid and sensitive isolation method. Yields of 14.0 ± 0.3 mg raw mitochondrial protein per 10⁸ cells were obtained. The mitochondria were morphologically intact, with membrane integrities of 67% (outer membrane) to 94% (inner membrane). Compared with the methods using Dounce homogenization, digitonin permeabilization, or electroporation for cell disruption the ultrasound method provided the highest yield of isolated mitochondria. Furthermore, this method is rapid (≈ 45 s for disruption), more robust than Dounce homogenization regarding their influence on mitochondrial integrity and especially suitable for preparing a relatively large amount of mitochondria. The results of this work can be helpful for quantitative and dynamic studies of molecular processes related to mitochondria under physiological conditions for many questions in both biomedicine and biotechnology.     

Isolation of functional pure mitochondria by superparamagnetic microbeads

Isolation of mitochondria by current methods relies mainly on their physicochemical properties. Here we describe an alternative approach to obtain functional mitochondria from human cells in a fast, reproducible, and standardized procedure. The new approach is based on superparamagnetic microbeads conjugated to anti-TOM22 antibody. The bead conjugates label the cytoplasmic part of the human mitochondrial membrane protein TOM22 and, thus, allow for a gentle isolation of mitochondria in a high gradient magnetic field. By comparing the MACS (magnetic cell separation) approach with mitochondria isolation methods using differential centrifugation and ultracentrifugation we demonstrate that the MACS approach provides the highest yield of isolated mitochondria. The quality, enrichment, and purity of mitochondria isolated with this protocol are comparable to mitochondria obtained using the ultracentrifuge method, and a typical separation procedure takes only approximately 1 to 2 h from initial cell homogenization. Mitochondria isolated with the new approach are sufficient for protein import, blue native gel electrophoresis, and other mitochondrial assays.     

Modified properties of hexokinase from heart mitochondria prepared using proteolytic enzyme

Summary Isolation of muscle mitochondria is made easier by using proteolytic treatment of the tissue before homogenization. Normally, the proteolytic enzyme is discarded with the supernatant of the first centrifugation. However, our results show that a fraction of enzyme activity remains associated with mitochondria. As shown in experiments described in this paper, mitochondrial hexokinase from tissue treated or not with the proteolytic enzyme exhibits similar properties except that the solubilized enzyme from protease treated tissue is no longer able to rebind to mitochondrial membrane. This modification of the binding ability of the enzyme results from a partial hydrolysis of hexokinase during solubilization experiments by the proteolytic enzyme. Since, as pointed out here, proteolytic enzyme can remain associated with mitochondria, [either adsorbed on mitochondrial membrane or included in the mitochondrial pellet] its use for the isolation of muscle mitochondria should be avoided.     

Isolation of mitochondria from rat brain using Percoll density gradient centrifugation

We have developed procedures that combine differential centrifugation and discontinuous Percoll density gradient centrifugation to isolate mitochondria from rat forebrains and brain subregions. The use of Percoll density gradient centrifugation is central to obtaining preparations that contain little contamination with synaptosomes and myelin. Protocols are presented for three variations of this procedure that differ in their suitability for dealing with large or small samples, in the proportion of total mitochondria isolated and in the total preparation time. One variation uses digitonin to disrupt synaptosomes before mitochondrial isolation. This method is well suited for preparing mitochondria from small tissue samples, but the isolated organelles are not appropriate for all studies. Each of the procedures produces mitochondria that are well coupled and exhibit high rates of respiratory activity. The procedures require an initial setup time of 45-75 min and between 1 and 3 h for the mitochondrial isolation.     

Adenosine 5'-monophosphate-removing system in fructose-1,6-bisphosphatase assay mixture: a new approach

An improved procedure for the isolation of mitochondria in high yields from normal and oxygen-deficient myocardium is described. The heart muscle is digested with Nagarse and homogenized simultaneously using a Polytron tissue homogenizer. Mitochondria are isolated by differential centrifugation, and othe subcellular fractions are carefully rinsed to maximize mitochondrial yields. Yields of 28 to 33 mg of mitochondrial protein/g wet wt of heart were obtained from normal (nonperfused and control perfused) hearts and from oxygen deficient (ischemic and autolyzed) hearts. This represents a recovery of 52 to 61% of the total mitochondrial content of the tissue. These mitochondria are functionally intact, with respiratory control ratios of 5.0 to 7.6 and $\text{ADP}\text{O}$ ratios of 2.34 to 2.66. The lysosomal content of the mitochondrial preparations was not increased by this procedure. This method is especially suitable for the preparation of mitochondria in high yield from a single heart, but can also be used to obtain high yields of mitochondria from larger quantities of myocardial tissue.     

Efficient Isolation of Pure and Functional Mitochondria from Mouse Tissues Using Automated Tissue Disruption and Enrichment with Anti-TOM22 Magnetic Beads

To better understand molecular mechanisms regulating changes in metabolism, as observed e.g. in diabetes or neuronal disorders, the function of mitochondria needs to be precisely determined. The usual isolation methods such as differential centrifugation result in isolates of highly variable quality and quantity. To fulfill the need of a reproducible isolation method from solid tissues, which is suitable to handle parallel samples simultaneously, we developed a protocol based on anti-TOM22 (translocase of outer mitochondrial membrane 22 homolog) antibody-coupled magnetic beads. To measure oxygen consumption rate in isolated mitochondria from various mouse tissues, a traditional Clark electrode and the high-throughput XF Extracellular Flux Analyzer were used. Furthermore, Western blots, transmission electron microscopic and proteomic studies were performed to analyze the purity and integrity of the mitochondrial preparations. Mitochondrial fractions isolated from liver, brain and skeletal muscle by anti-TOM22 magnetic beads showed oxygen consumption capacities comparable to previously reported values and little contamination with other organelles. The purity and quality of isolated mitochondria using anti-TOM22 magnetic beads was compared to traditional differential centrifugation protocol in liver and the results indicated an obvious advantage of the magnetic beads method compared to the traditional differential centrifugation technique.     

Maize mitochondria: purification and characterization of ribosomes and ribosomal ribonucleic Acid

Mitochondria were prepared from etiolated maize shoots (Zea mays L. var. McNair 508) by homogenization followed by differential centrifugation and equilibrium banding in discontinuous sucrose or Renografin-sucrose gradients. Mitochondria prepared by sucrose banding showed better physiological integrity than those prepared by renografin-sucrose banding, although both procedures yielded mitochondria that showed respiratory control and coupling of oxidation to phosphorylation of ADP. Mitochondria prepared by Renografin-sucrose banding were free of dectectable cytoplasmic ribosomal RNA, while sucrose banding resulted in a low level of contamination. Ribosomes isolated from mitochondria sedimented at about 78S, with subunits sedimenting at 60 and 44S. Using Escherichia coli ribosomal RNA as internal standards, the molecular weights of mitochondrial ribosomal RNAs were found to be 0.74 to 0.75 and 1.26 × 10⁶ daltons by polyacrylamide gel electrophoresis, before or after denaturation in formaldehyde. Cytoplasmic ribosomal RNA molecular weights were 0.70 and 1.26 × 16⁶ before denaturation, and 0.68 and 1.5 × 10⁶ after denaturation, suggesting an unusual reaction of the heavy ribosomal RNA to formaldehyde.     

Hydrodynamic parameters and isolation of mitochondria, microperoxisomes and microsomes of rat heart

1. Analytical differential centrifugation of rat heart homogenates revealed a single population of mitochondria and microperoxisomes. Using cytochorme c oxidase, malate dehydrogenase and amine oxidase as mitochondrial marker enzymes, the s̄-value of mitochondria was estimated to s̄ = 10326 ± 406 S (average for the three marker enzymes). The −s-value of microperoxisomes was found to be −s = 1381 ± 40 S using catalase as the marker enzyme. The −s-value for the two orgenelles did not change significantly when the isoosmotic sucrose medium was substituted by an isoosmotic mannitol medium. 2. Analytical differential centrifugation revealed a polydispercity of the microsomal fraction using glucose-6-phosphatase and NADPH-cytochrome c reductase as the marker enzymes. The s̄-values were found to be −s_H1 = 1569 ± 412 S (NADPH-cytochrome c reductase), (glucose-6-phosphatase) and (NADPH-cytochrome c reductase and glucose-6-phosphatase). The recovery of marker enzymes in the isolated subcellular fractions was in the range of 84–94%. 3. When the mitochondrial and microperoxisomal fractions were subjected to isopycnic gradient centrifugation, using a self-generating gradient of polyvinylpyrrolidone-coated colloidal silica particles (Percoll) in 0.25 M sucrose medium, buoyant densities of 1.10 g/cm³ (main fraction of mitochondria) and 1.06 g/cm³ (main fraction of microperixosomes) were obtained. The density gradient centrifugation separated microperoxisomes from contaminating lysosomes of high specific activity in acid phosphatase. A value 1.04 g/cm³ was foung for the density of the microsomal fraction. 4. Based on the estimated -values, an optimal procedure is described for the isolattion of mitochondrial and microperoxisomal fractions from rat heart muscle.     

Isolation and reconstruction of cardiac mitochondria from SBEM images using a deep learning-based method

Mitochondrial morphological defects are a common feature of diseased cardiac myocytes. However, quantitative assessment of mitochondrial morphology is limited by the time-consuming manual segmentation of electron micrograph (EM) images. To advance understanding of the relation between morphological defects and dysfunction, an efficient morphological reconstruction method is desired to enable isolation and reconstruction of mitochondria from EM images. We propose a new method for isolating and reconstructing single mitochondria from serial block-face scanning EM (SBEM) images. CDeep3M, a cloud-based deep learning network for EM images, was used to segment mitochondrial interior volumes and boundaries. Post-processing was performed using both the predicted interior volume and exterior boundary to isolate and reconstruct individual mitochondria. Series of SBEM images from two separate cardiac myocytes were processed. The highest F1-score was 95% using 50 training datasets, greater than that for previously reported automated methods and comparable to manual segmentations. Accuracy of separation of individual mitochondria was 80% on a pixel basis. A total of 2315 mitochondria in the two series of SBEM images were evaluated with a mean volume of 0.78 µm³. The volume distribution was very broad and skewed; the most frequent mitochondria were 0.04–0.06 µm³, but mitochondria larger than 2.0 µm³ accounted for more than 10% of the total number. The average short-axis length was 0.47 µm. Primarily longitudinal mitochondria (0–30 degrees) were dominant (54%). This new automated segmentation and separation method can help quantitate mitochondrial morphology and improve understanding of myocyte structure–function relationships.     

Simple procedure for rapid isolation of functionally intact mitochondria from human and rat skeletal muscle

A simple procedure for the rapid isolation of functionally intact skeletal muscle mitochondria is described. The method involves homogenization of muscle in a medium comprising sucrose (0.25 M) containing 50,000 units of heparin/liter, followed by differential centrifugation. Mitochondria so isolated are functionally and morphologically intact.     

The aromatization of cyclohexanecarboxyl-CoA to hippuric acid by guinea pig liver mitochondria: submitochondrial localization

Summary The conversion of cyclohexanecarboxyl-CoA to hippuric acid in submit ochondrial fractions from guinea pig liver was studied using a gas chromatographic-mass spectrometric method employing selected ion monitoring. Comparison of the activities of the cyclohexanecarboxyl-CoA to hippuric acid converting system (CCoAHC-system) and marker enzymes in the various submit ochondrial fractions showed that the CCoAHC-system is localized in the mitochondrial matrix. Partial separation of the inner and outer membranes has been accomplished by treating mitochondria with digitonin in isotonic medium and fractionating the treated mitochondria by differential centrifugation. A digitonin-protein ratio of 2.6 mg of digitonin/10 mg of protein must be used in order to release significant amounts of amine oxidase activity (outer membrane marker) from low speed mitochondrial pellets. This pellet still contained most of the glutamate dehydrogenase activity and was insignificantly contaminated with adenylate kinase. Moderate concentrations of phenazine methosulfate (PMS) greatly stimulated the activity of the CCoAHC-system, even in intact mitochondria (optimal concentration of PMS: 1 mM) whilst higher concentrations (> 1 mM) decreased the activity. The formation of hippuric acid in these mitochondrial preparations was linear with time for at least 40 min and linear with respect to protein concentration up to approximately 2.0 mg mitochondrial protein·m¹.     

Comparative analyses of cell disruption methods for mitochondrial isolation in high-throughput proteomics study

One of the most crucial steps in mitochondrial isolation is disruption of intact cells to denude intracellular organelles, but the yield and purity of different disruption protocols have not been well addressed. In the present study, MDCK cells were disrupted by mechanical (sonication and homogenization), physical (repeated freeze/thaw cycles and hypoosmotic burst), and chemical (using Triton X-100, NP-40, or CHAPS) methods. Efficacy of cell disruption was evaluated by trypan blue staining and mitochondria were subsequently isolated by standardized differential centrifugation. The yield of isolation was also determined by measuring protein concentrations, whereas the purity was examined by Janus green B staining, Western blot analyses of markers for mitochondria (COX-4) and other subcellular organelles/locales (i.e., nucleus, cytoplasm, endoplasmic reticulum, and lysosome), transmission electron microscopy, two-dimensional electrophoresis, and Q-TOF MS and/or MS/MS analyses. Our data demonstrated that sonication is the method of choice for disruption of cells prior to mitochondrial isolation for proteome analysis.     

The isolation of intact adrenal chromaffin granules using isotonic Percoll density gradients

An improved method for the isolation of intact adrenal chromaffin granules under isotonic conditions, using a Percoll density gradient, is presented. After dissection, homogenization, and differential centrifugation, the crude granule homogenate was layered onto a gradient medium previously centrifuged at 20,200g × 5 min, consisting of 30% ($\text{v}\text{v}$) Percoll, 0.27 m sucrose, and 10 mm Tris-maleate, pH 7.0. After centrifugation for 40 min at 8650g in a standard preparative centrifuge, the chromaffin granules were found to band in the lowest fraction, where 45% of the catecholamines and 60% of the ATP could be recovered. With respect to other published methods, the percentage of lysosomal and mitochondrial contamination compared favorably. In addition, granules isolated by the Percoll gradient method were found to have at least 42 and 14% higher ATP and catecholamines, respectively, per milligram of protein. It is suggested that this method offers the advantages of ease of preparation, purity, and cost efficiency when compared with previously published techniques.     

A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM)

This protocol details methods for the isolation of yeast nuclei from budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), immuno-gold labeling of proteins and visualization by field emission scanning electron microscopy (FESEM). This involves the removal of the yeast cell wall and isolation of the nucleus from within, followed by subsequent processing for high-resolution microscopy. The nuclear isolation step can be performed in two ways: enzymatic treatment of yeast cells to rupture the cell wall and generate spheroplasts (cells that have partially lost their cell wall and their characteristic shape), followed by isolation of the nuclei by centrifugation or homogenization; and whole cell freezing followed by manual cell rupture and centrifugation. This protocol has been optimized for the visualization of the yeast nuclear envelope (NE), nuclear pore complexes (NPCs) and associated cyto-skeletal structures. Samples once processed for FESEM can be stored under vacuum for weeks, allowing considerable time for image acquisition.     

ISOLATION AND CHARACTERIZATION OF THE SARCOPLASMIC RETICULUM OF SKELETAL MUSCLE

The sarcoplasmic reticulum (SR) of rabbit skeletal muscle was studied after isolation of a vesicle fraction and of vesicular subfractions by means of differential and density gradient centrifugations. The different fractions were examined electron microscopically by negative and positive staining; their content in protein and phospholipid and their ability to bind Ca⁺⁺ were determined. After homogenization, differential centrifugation yielded a "sarcovesicular fraction" (SVF) which was mainly composed of numerous vesicles of different types mixed with fibrous proteins and mitochondrial fragments. This SVF contained 2% of the protein and 25% of the phospholipid of the initial tissue extract. It had a high Ca⁺⁺ binding activity that was preserved for several days by storage in the presence of oxalate. After centrifugations of the SVF on sucrose density gradients, two vesicular subfractions were obtained which were characterized by different sedimentation rates, isopycnic banding, morphology, and composition in protein and phospholipid. (a) The low-density subfraction (ρ 1.10–1.12) contained a heterogeneous population of membranous structures: thick- and thin-walled vesicles, tubular formations, triads, and plasma membranes. Its content in protein and phospholipid was very low. (b) The high-density subfraction (ρ 1.13–1.17) was a very pure subfraction composed only of thin-walled vesicles. Its content in phospholipid was high and the ratio of phospholipid-phosphorus to protein was about 20. The calcium-binding activity found in the total SVF was recovered only in this latter homogeneous subfraction. The origin of these two subfractions from the SR is discussed.     

Localization of [14C]DDT in the ventral nerve cord of the cockroach,Periplaneta americana (L.)

[¹⁴C]DDT was used as a probe to determine the subcellular localization of DDT in the ventral nerve cord (VNC) of the cockroach, Periplaneta americana (L.). Male cockroaches were injected intra-abdominally with [¹⁴C]DDT and their VNCs removed at 1 h post-injection. The VNCs were then subjected to homogenization and differential centrifugation to isolate plasma membrane, mitochondrial, and microsomal fractions. Results indicate that the plasma membrane fraction contained the greatest amount of [¹⁴C]DDT, with the mitochondrial and microsomal fractions containing significantly less. Calculations and a comparison with I₅₀ values for oligomycin-sensitive (OS)Mg-ATPase from the literature support the prediction that an insufficient amount of DDT reaches the ventral nerve cord mitochondria of a cockroach to effect an I₅₀ level of inhibition of the (OS)Mg-ATPase.     

Heterogeneity of chondrocyte mitochondria: A study of the Ca concentration and density banding characteristics of normal and rachitic cartilage

Previous morphological studies of the mineralizing epiphysis suggested that some mitochondria were concerned with Ca²⁺ accumulation while others were associated with cellular energetics and metabolism. To determine if there was mitochondrial heterogeneity in chondrocytes of the epiphyseal growth plate, mitochondria were isolated from four different regions of the plate and subjected to continuous sucrose gradient centrifugation. Centrifugation of the organelles in a narrow density sucrose gradient (1.5–2.0 M) in the presence of inhibitors of Ca²⁺ transport (ruthenium red and 5,5′-dithiobis-(2-nitrobenzoic acid)) revealed that considerable heterogeneity existed. In the least calcified zone 20% of the mitochondria formed a low density band of low Ca²⁺ concentration (309 nmol/mg protein). Organelles isolated from more calcified tissue zones showed a concomitant increase in Ca²⁺ concentration (up to 5700 nmol/mg protein) as well as an increase in the total percentage of mitochondria sedimenting in 2.0 M sucrose. The banding patterns of mitochondria isolated from rachitic and hypertrophic cartilage were similar. In addition, similarities were also noted in the Ca²⁺ concentration and the cytochrome oxidase activities of mitochondria of these tissues. During recovery from the rachitic condition, there was a change in the density centrifugation characteristics of this tissue and a substantial increase was noted in the proportion of mitochondria sedimenting in 2.0 M sucrose. The Ca²⁺ concentration of mitochondria of this rapidly calcifying tissue suggested that the critical Ca²⁺ concentration necessary for initiation of the calcification mechanism was 4 μmol/mg protein.     

A simple method for the large-scale preparation of mitochondria from microorganisms.

A simple mechanical procedure that has been developed for the large-scale preparation of intact mitochondria from yeast, is also applicable to the extraction of organelles from other organisms having cell walls. A procedure for the isolation of large quantities of pure mitochondrial DNA from these mitochondria is described. In Schizosaccharomyces pombe, further purification of the mitochondria by urografin isopycnic centrifugation leads to 50% recovery of whole cell respiration activity in a vesicular fraction of respiratory chain enzymes, with NADH oxidase activity usually greater than 10 μmol of electrons/min/mg of protein. The method has the advantage of rapidity and low cost and it is extremely healthy for the operator.     

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 μg of mitochondria indicate that 1.0 μg is sufficient to measure respiration and that 5.0 μg provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.