Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator’s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 10¹⁰ viable and respiration competent mitochondria from 0.18 ± 0.04 g (wet weight) tissue sample.     

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.     

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 and thin-layer chromatographic identification of several peptides with NH2-terminal tryptophan and their histochemical demonstration in the ACTH cells of the rat hypophysis

Methods for the isolation and thin-layer chromatographic identification of amino-terminal tryptophyl-peptides presumably responsible for histochemical tryptophyl-peptide reactions in the ACTH cells of the rat hypophysis are described. In the hypophyseal extract several tryptophylpeptide bands--depending on the homogenization solution--were demonstrated on thin-layer chromatograms. Tryptophyl-peptides were demonstrated from their fluorescence induced 1) with glyoxylic acid (glyoxylic acid introduced into the homogenization solution), 2) by exposure of the chromatographic plates to combined formaldehyde and chloral vapour or 3) by exposure to combined formaldehyde and acetyl chloride vapour. A positive PAS reaction was demonstrated in some tryptophyl-peptide bands. Thus, some tryptophylpeptides seem to contribute to the observed PAS positivity of the ACTH cells.     

Isolation of intact mitochondria and hepatocytes using vibration

A method of isolation of rat liver cells and mitochondria suggesting tissue disaggregation by vibration has been proposed. Mitochondria obtained using vibration have better parameters of oxidative phosphorylation as compared to homogenization. Hepatocytes isolated by vibration had viability about 90%, as determined by trypan blue exclusion, and rates of respiration and xenobiotic metabolism comparable to that observed by the enzymatic method.     

A new method for the isolation of plasma membranes from amphibian embryos

Summary A method for the isolation of plasma membranes is described. N-Succinimidyl 3-(2-pyridyldithio) propionate (SPDP), a heterobifunctional reagent, is covalently linked to protein amino groups in plasma membranes of intact cells. After homogenization of the cells the plasma membranes can be separated from other cell components by selective coupling to reduced Thiopropyl-Sepharose 6B and then recovered after treatment with 2-mercaptoethanol.     

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.     

High throughput preparation of fly genomic DNA in 96-well format using a paint-shaker

Sample homogenization is an essential step for genomic DNA extraction, with multiple downstream applications in Molecular Biology. Genotyping hundreds or thousands of samples requires an automation of this homogenization step, and high throughput homogenizer equipment currently costs 7000 euros or more. We present an apparatus for homogenization of individual Drosophila adult flies in 96-well micro-titer dishes, which was built from a small portable paint-shaker (F5 portable paint-shaker, Ushake). Single flies are disrupted in each well that contains extraction buffer and a 4-mm metal ball. Our apparatus can hold up to five 96-well micro-titer plates. Construction of the homogenizer apparatus takes about 3–4 days, and all equipment can be obtained from a home improvement store. The total material cost is approximately 700 euros including the paint-shaker. We tested the performance of our apparatus using the ZR-96 Quick-gDNA? kit (Zymo Research) homogenization buffer and achieved nearly complete tissue homogenization after 15 minutes of shaking. PCR tests did not detect any cross contamination between samples of neighboring wells. We obtained on average 138 ng of genomic DNA per fly, and DNA quality was adequate for standard PCR applications. In principle, our tissue homogenizer can be used for isolation of DNA suitable for library production and high throughput genotyping by Multiplexed Shotgun Genotyping (MSG), as well as RNA isolation from single flies. The sample adapter can also hold and shake other items, such as centrifuge tubes (15–50 mL) or small bottles.     

Isolation and thin-layer chromatographic identification of several peptides with NH2-terminal tryptophan and their histochemical demonstration in the ACTH cells of the rat hypophysis

Summary Methods for the isolation and thin-layer chromatographic identification of amino-terminal tryptophyl-peptides presumably responsible for histochemical tryptophyl-peptide reactions in the ACTH cells of the rat hypophysis are described. In the hypophyseal extract several tryptophylpeptide bands — depending on the homogenization solution — were demonstrated on thin-layer chromatograms. Tryptophyl-peptides were demonstrated from their fluorescence induced 1) with glyoxylic acid (glyoxylic acid introduced into the homogenization solution), 2) by exposure of the chromatographic plates to combined formaldehyde and chloral vapour or 3) by exposure to combined formaldehyde and acetyl chloride vapour. A positive PAS reaction was demonstrated in some tryptophyl-peptide bands. Thus, some tryptophyl-peptides seem to contribute to the observed PAS positivity of the ACTH cells.This investigation was supported by grants from the Jalmari and Rauha Ahokas Foundation and the J.K. Paasikivi Foundation     

Reversible inactivation by noradrenaline of long-chain fatty acyl-CoA synthetase in rat adipocytes.

Incubation of rat adipocytes with the same range of noradrenaline concentrations that stimulate lipolysis caused a rapid and stable decrease in the activity of fatty acyl-CoA synthetase. Corticotropin, glucagon and dibutyryl cyclic AMP also decreased the activity of the enzyme. The effect of noradrenaline was apparent over a wide range of concentrations for the three substrates of the enzyme. A novel fluorescence assay of fatty acyl-CoA synthetase using (1,N6-etheno)-CoA is described. The effect of noradrenaline was not abolished by inclusion of albumin in homogenization buffers, persisted through subcellular fractionation and isolation of microsomes (microsomal fractions) and even survived treatment of microsomes with Triton X-100. The effect of noradrenaline was rapidly reversed within cells by the subsequent addition of insulin or propranolol. The inclusion of fluoride in homogenization buffers did not alter the observed effect of noradrenaline. Additions of cyclic AMP-dependent protein kinase to adipocyte microsomes caused considerable phosphorylation of microsomal protein by [gamma-32P]ATP, but did not affect the activity of fatty acyl-CoA synthetase.     

Efficacy of various pasteurization time-temperature conditions in combination with homogenization on inactivation of Mycobacterium avium subsp. paratuberculosis in milk

The effect of various pasteurization time-temperature conditions with and without homogenization on the viability of Mycobacterium avium subsp. paratuberculosis was investigated using a pilot-scale commercial high-temperature, short-time (HTST) pasteurizer and raw milk spiked with 10(1) to 10(5) M. avium subsp. paratuberculosis cells/ml. Viable M. avium subsp. paratuberculosis was cultured from 27 (3.3%) of 816 pasteurized milk samples overall, 5 on Herrold's egg yolk medium and 22 by BACTEC culture. Therefore, in 96.7% of samples, M. avium subsp. paratuberculosis had been completely inactivated by HTST pasteurization, alone or in combination with homogenization. Heat treatments incorporating homogenization at 2,500 lb/in2, applied upstream (as a separate process) or in hold (at the start of a holding section), resulted in significantly fewer culture-positive samples than pasteurization treatments without homogenization (P < 0.001 for those in hold and P < 0.05 for those upstream). Where colony counts were obtained, the number of surviving M. avium subsp. paratuberculosis cells was estimated to be 10 to 20 CFU/150 ml, and the reduction in numbers achieved by HTST pasteurization with or without homogenization was estimated to be 4.0 to 5.2 log10. The impact of homogenization on clump size distribution in M. avium subsp. paratuberculosis broth suspensions was subsequently assessed using a Mastersizer X spectrometer. These experiments demonstrated that large clumps of M. avium subsp. paratuberculosis cells were reduced to single-cell or "miniclump" status by homogenization at 2,500 lb/in2. Consequently, when HTST pasteurization was being applied to homogenized milk, the M. avium subsp. paratuberculosis cells would have been present as predominantly declumped cells, which may possibly explain the greater inactivation achieved by the combination of pasteurization and homogenization.     

Production of immobilized penicillin acylase using aqueous polymer systems for enzyme purification and in situ immobilization

A novel approach for the isolation and purification of penicillin acylase (PA), which couples aqueous two-phase partitioning and enzyme immobilization has been investigated.A PA yield of 90% was achieved by treating E. coli cells with 4% butyl acetate, freeze-thawing step, and pressure homogenization. PA purification (93% recovery) was achieved by (1) removing cell debris via precipitation with polyethylene glycol (PEG 2000); (2) aqueous two-phase partitioning using a PEG 2000 + phosphate system (87% recovery).An in situ enzyme immobilization approach, using oxirane acrylic or aldehyde-agarose beads dispersed in the PEG-rich phase, was explored for the conversion of penicillin G to 6-aminopenicillanic acid. An appropriate immobilization reaction time was found. The catalytic performance of the enzyme, when immobilized, was found not to be affected by recycling of the phase-forming components.     

Islands in cities: Urbanization and fragmentation drive taxonomic and functional variation in ground arthropods

The conversion of natural lands in urban areas is exponentially increasing worldwide, causing a major decline in biodiversity. Environmental alterations caused by urbanization, such as land conversion and isolation of natural patches, favour tolerant and generalist species, causing both species loss and replacement. In addition, selective pressure is exerted on particular functional traits, driving a functional homogenization or turnover of biotic communities. We sampled ground arthropods within the municipality of Turin (NW-Italy), wherein an isolated and a connected control subplot were repeatedly sampled at 15 stations distributed along a gradient of increasing urbanization. Such a nested sampling design allowed us to investigate the taxonomic and the functional responses of carabids and spiders to both the urbanization level and patch isolation. First, we highlighted the dominant role played by species homogenization (nestedness) in explaining both taxonomic and functional variation in both groups of arthropods. Secondly, we showed that urbanization causes simultaneously functional homogenization and replacement in both carabid and spider assemblages, whereas patch isolation influences carabid species composition and homogenizes and shifts spider taxonomic and functional composition. Lastly, by relating community-weighted means of body length, dispersal capacity and trophic strategy to the urbanization and isolation gradients, we demonstrated that urbanization alters the trophic structure of both taxonomic groups and increases the average dispersal capacity of spiders. On the other hand, patch isolation affected the functional composition of spiders only, reducing the body size and increasing dispersal capacity and the proportion of web-builder species. Our results demonstrate that both urbanization and patch isolation alter species composition by causing functional and taxonomic homogenization. In addition, they exert a strong filtering effect on community functional traits, increasing the proportion of phytophagous species in carabids, and increasing dispersal capacity and web-builders occurrence in spiders, while reducing spider body size.     

High throughput preparation of fly genomic DNA in 96-well format using a paint-shaker

Sample homogenization is an essential step for genomic DNA extraction, with multiple downstream applications in Molecular Biology. Genotyping hundreds or thousands of samples requires an automation of this homogenization step, and high throughput homogenizer equipment currently costs 7000 euros or more. We present an apparatus for homogenization of individual Drosophila adult flies in 96-well micro-titer dishes, which was built from a small portable paint-shaker (F5 portable paint-shaker, Ushake). Single flies are disrupted in each well that contains extraction buffer and a 4-mm metal ball. Our apparatus can hold up to five 96-well micro-titer plates. Construction of the homogenizer apparatus takes about 3–4 days, and all equipment can be obtained from a home improvement store. The total material cost is approximately 700 euros including the paint-shaker. We tested the performance of our apparatus using the ZR-96 Quick-gDNA™ kit (Zymo Research) homogenization buffer and achieved nearly complete tissue homogenization after 15 minutes of shaking. PCR tests did not detect any cross contamination between samples of neighboring wells. We obtained on average 138 ng of genomic DNA per fly, and DNA quality was adequate for standard PCR applications. In principle, our tissue homogenizer can be used for isolation of DNA suitable for library production and high throughput genotyping by Multiplexed Shotgun Genotyping (MSG), as well as RNA isolation from single flies. The sample adapter can also hold and shake other items, such as centrifuge tubes (15–50 mL) or small bottles.     

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.     

An improved Method for Isolation of Rat Testis Golgi Apparatus H. H. Mollenhauer

The preparation of Golgi apparatus fractions from rat testis germ cells free from contamination by residual body fragments was accomplished by the use of the Yeda press as the homogenization device. The Golgi apparatus thus prepared retained excellent stuctural intactness. This method also allows for isolation of Golgi apparatus from single cell suspensions.     

A rapid procedure for isolating hemopoietic cell nuclei

A new method for isolating cell nuclei is described which involves freezing and thawing cells in 2% Tween 40, then gentle homogenization to release nuclei, followed by immediate microcentrifugation through 50% sucrose. Purified nuclei were obtained in 3 min and yields of 78-95% were obtained from a variety of human hemopoietic cells. Electron microscope analysis of nuclei obtained from HL60 cells showed that 89% of the nuclei were intact and have an appropriate morphology. A low level of contamination with other organelles was revealed by electron microscopy and by using specific assays for plasma membrane, mitochondria, lysosomes, Golgi membrane, and endoplasmic reticulum (0.5-5.5%). The value of the technique is that nuclear proteins and small metabolites which might be lost by rapid leakage from isolated nuclei and the possibility of biochemical modification of cellular constituents are minimized by using a rapid isolation procedure.     

Enucleation of human endometrial cells: nucleo-cytoplasmic distribution of DNA polymerase alpha and estrogen receptor

Nucleo-cytoplasmic distribution of estrogen receptors and DNA polymerase alpha activity in human endometrial adenocarcinoma cells (HEC-50 line) was evaluated after separation of nuclei following either homogenization or enucleation with cytochalasin B. About 30% of the estrogen receptor was found in the nuclear fraction after homogenization whereas 86% was found in the karyoplasts after enucleation. The total amounts of estrogen receptor per cell after homogenization and enucleation were not significantly different (14,000-17,000 binding sites/cell). Receptor measurements were carried out using the hydroxylapatite method after labeling with [3H]estradiol (5 nM [3H]E2 +/- 500 nM E2) at 30 degrees C for 3 h. About 20% of the DNA polymerase alpha activity was found in the nuclear fraction after homogenization, whereas 96% was found in the karyoplasts after enucleation. The average total activity (0.84 Units/10(6) cells) in homogenized cells was about 1/8 of the activity in karyoplasts. These results indicate that estrogen receptor and DNA polymerase alpha activity reside in the nucleus in intact HEC-50 cells. DNA polymerase alpha is translocated to the cytoplasmic fraction and inactivated after homogenization.     

Cumulative sedimentation analysis of Escherichia coli debris size

A new method to measure Escherichia coli cell debris size after homogenization is presented. It is based on cumulative sedimentation analysis under centrifugal force, coupled with Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis of sedimented proteins. The effects that fermentation and homogenization conditions have on the resulting debris distributions were investigated using this method. Median debris size decreased significantly from approximately 0.5 mum to 0.3 mum as the number of homogenization passes increased from 2 to 10. Under identical homogenization conditions, uninduced host cells in stationary phase had a larger debris size than exponential cells after 5 homogenizer passes. This difference was not evident after 2 or 10 passes, possibly because of confounding intact cells and the existence of a minimum debris size for the conditions investigated. Recombinant cells containing protein inclusion bodies had the smallest debris size following homogenization. The method was also used to measure the size distribution of inclusion bodies. This result compared extremely well with an independent determination using centrifugal disc photosedimentation (CDS), thus validating the method. This is the first method that provides accurate size distributions of E. coli debris without the need for sample pretreatment, theoretical approximations (e.g. extinction coefficients), or the separation of debris and inclusion bodies prior to analysis. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioang 55: 556-564, 1997.     

Different methods of membrane domains isolation result in similar 2-D distribution patterns of membrane domain proteins.

Membrane domains are highly specialized parts of the cell plasma membrane, carrying on and augmenting the incoming signals. To study their structural and functional properties, it is crucial to find the least damaging mode of their isolation. Using two different cell lines, epithelial HEK cells (clone E2M11) and S49 lymphoma cells, three methods of membrane domain isolation (i.e., detergent extraction, alkaline treatment, and "drastic" homogenization) were tested for similarity and reproducibility by 2-D electrophoresis. Our data show that the protein composition of membrane domains obtained by different isolation methods is similar and that approximately 60% of the spots are present in all membrane domain preparations. Furthermore, the same degree of similarity of 2-D profiles of the most intensively silver stained spots found in membrane domains of the two cell lines derived from different tissues suggests that the composition of a large part of membrane domains proteins is conservative. We suggest that these proteins may either be involved in the organization of membrane domain structure or represent the conservative component of signal transduction machinery.