cellular debris magnetic separator on the site

For the original ore, sinter, pellets, lump ore and other magnetic ore separator on the conveyor belt. HTK system relies on magnetic iron ore in addition to its unique magnetic circuit design of the selected materials for many separation, which will ferromagnetic debris in the way with the least amount of ore minerals from the magnetic compound sucked, to protect the continued purpose of crushing equipment.

5.Automatic unloading iron, easy maintenance, drum drum-type structure, with auto-correction feature, specially sealed bearing to meet the on-site dust big occasions, can achieve long-term trouble-free operation.

Right now,Tianli have developed more than thirty series of Tianli brand magnetic separators,Deferrization magnets,and mining equipments,etc.which include hundreds of products with different specifications.Tianli is good reputaion magnet supplier,magnetic separator supplier in china.If you are looking for the quality magnetic separator vendor,magnetic separator distributor,magnetic separatorfactory,or magnetic separator company. Tianli is your good choice.

slon seperator - billinton group

slon seperator - billinton group

Billinton Group Pty. Limited (BG) and SLon Magnetic Separator Ltd (SLMS) from China, have established a strategic cooperation relationship regarding to the business of SLon magnetic separators. BG are authorized to market and sell, in Australia and the Pacific Region, SLMS's series of SLon magnetic separation products equipped with the world leading technology, having an absolutely dominant position in China's market for beneficiation of low grade iron ore. Any iron ore exploration and mining company in Australia, especially those mining developers involved low grade iron ore (hematite, limonite, ilmenite, manganese), and those who have a desire to upgrade their low grade iron ore to 60% or even 65%, please contact us eitherThis email address is being protected from spambots. You need JavaScript enabled to view it.orThis email address is being protected from spambots. You need JavaScript enabled to view it., we are glad to cooperate with you.

Slon Magnetic Separator Ltd has successfully developed a new magnetic separator, SLon-4000 vertical ring pulsating high gradient magnetic separator, a machine having the largest processing capacity of a single unit ever in China. This machine was installed and successfully run in an iron mine in China in November, 2011. The technical parameters meet or exceed the design requirements.

The machine has a swivel ring of outer diameter as large as 4 meters, weighs nearly 400 tons and is capable of processing 550 tons of ore per hour. It is the largest single processing capability machine among the magnetic separators applied in China and in the world. The success of development, installation and operation of this machine is a landmark to large scale mineral processing equipment in China and even in the world.

According to the customers' requirements and ore types and properties, we will carry out detailed laboratory testwork, testifying whether the ores are capable of being separated, determining the optimal conditions for separation and selecting the most appropriate type of equipment. We can also design a special machine to be suitable to the users' special requirements.

Provide all SLon products installation free of charge, and a long-term technical support. We are equipped with a team of hard-working and very experienced engineers who have all been working over the world for many years with the magnetic separators, providing related technical services, guiding installation and on-site-commissioning and training in operation and maintenance of the equipment.

Provide a long term supply of components and parts. Each machine is strictly conducted in inspection and test with electrical field, running-up and other quality testing procedures before it is sent out to ensure a 100% qualified product. The equipment and main parts are entitled to one year warranty (excluding wearing parts and consumables).

Tips: For a small laboratory test, please mail five (5) kilograms of representative samples. Please also tell us your ore properties and testing requirements. We will carry out laboratory testwork, testify whether the samples are separable, and determine the optimal conditions for separation. The test results will be timely informed to you. Please contact us where and how to mail the samples.

Please contact eitherThis email address is being protected from spambots. You need JavaScript enabled to view it.orThis email address is being protected from spambots. You need JavaScript enabled to view it.now.

Note: After the power is off the machine has to be running at idle for 2 minutes or more until those magnetic materials accumulated with high magnetic filed have been washed out before permitted to shut off the water and turn off the machine.

magnetic particles for the separation and purification of nucleic acids | springerlink

magnetic particles for the separation and purification of nucleic acids | springerlink

Nucleic acid separation is an increasingly important tool for molecular biology. Before modern technologies could be used, nucleic acid separation had been a time- and work-consuming process based on several extraction and centrifugation steps, often limited by small yields and low purities of the separation products, and not suited for automation and up-scaling. During the last few years, specifically functionalised magnetic particles were developed. Together with an appropriate buffer system, they allow for the quick and efficient purification directly after their extraction from crude cell extracts. Centrifugation steps were avoided. In addition, the new approach provided for an easy automation of the entire process and the isolation of nucleic acids from larger sample volumes. This review describes traditional methods and methods based on magnetic particles for nucleic acid purification. The synthesis of a variety of magnetic particles is presented in more detail. Various suppliers of magnetic particles for nucleic acid separation as well as suppliers offering particle-based kits for a variety of different sample materials are listed. Furthermore, commercially available manual magnetic separators and automated systems for magnetic particle handling and liquid handling are mentioned.

Magnetic separation is an emerging technology that uses magnetism for the efficient separation of micrometre-sized para- and ferromagnetic particles from chemical or biological suspensions. Enrichment of low-grade iron ore, removal of ferromagnetic impurities from large volumes of boiler water in both conventional and nuclear power plants, or the removal of weakly magnetic coloured impurities from kaolin clay are typical examples of magnetic separation in traditional industries. The application of these techniques in biosciences had been restricted and of limited use up to the 1970s. The idea of using magnetic separation techniques to purify biologically active compounds (nucleic acids, proteins, etc.), cells, and cell organelles led to a regrowing interest over the last decade. New magnetic particles with improved properties were developed for the partly complicated separation processes in these fields [see reviews: Olsvik et al. 1994; Safarik and Safarikova 1999; Franzreb et al. 2006].

Magnetic separation of nucleic acids has several advantages compared to other techniques used for the same purpose. Nucleic acids can be isolated directly from crude sample materials such as blood, tissue homogenates, cultivation media, water, etc. The particles are used in batch processes where there are hardly any restrictions with respect to the sample volumes. Due to the possibility of adjusting the magnetic properties of the solid materials, they can be removed relatively easily and selectively even from viscous sample suspensions. In fact, magnetic separation is the only feasible method for the recovery of small particles (diameter approx. 0.051m) in the presence of biological debris and other fouling material of similar size. Furthermore, the efficiency of magnetic separation is especially suited for large-scale purifications (Safarik et al. 2001; Franzreb et al. 2006).

These upcoming separation techniques also serve as a basis of various automated low- to high-throughput procedures that allow to save time and money. Centrifugation steps can be avoided and the risk of cross-contamination when using traditional methods is no longer encountered. Various types of magnetic particles are commercially available for nucleic acid purification, magnetic separators working in the manual and automated mode are offered. A short description of traditional and magnetic separation methods for nucleic acid isolation, together with a short overview of batch and automated separators, will be given below.

The isolation of DNA or RNA is an important step before many biochemical and diagnostic processes. Many downstream applications such as detection, cloning, sequencing, amplification, hybridisation, cDNA synthesis, etc. cannot be carried out with the crude sample material. The presence of large amounts of cellular or other contaminating materials, e.g. proteins or carbohydrates, in such complex mixtures often impedes many of the subsequent reactions and techniques. In addition, DNA may contaminate RNA preparations and vice versa. Thus, methods for the efficient, reliable and reproducible isolation of nucleic acids from complex mixtures are needed for many methods that are used today and rely on the identification of DNA or RNA, e.g. diagnosis of microbial infections, forensic science, tissue and blood typing, detection of genetic variations, etc.

A range of methods are known for the isolation of nucleic acids in the fluid phase, but they are generally based on complex series of precipitation and washing steps and are time-consuming and laborious to perform. Thus, classical methods for the isolation of nucleic acids from complex starting materials such as blood or tissues, involve the lysis of the biological material by a detergent or chaotropic substance, possibly in the presence of protein-degrading enzymes, followed by several processing steps applying organic solvents such as phenol and/or chloroform or ethanol, which in general are highly toxic and require special and, hence, expensive disposal. For example, the complete removal of proteins from nucleic acids can be achieved by the addition of sodium perchlorate (Wilcockson 1973). The separation of RNA from DNA requires selective precipitation steps with LiCl or a specific nuclease-free isolation with guanidinium hydrochloride or guanidinium thiocyanate, combined with phenol extraction and ethanol precipitation (Bowtell 1987). Such methods are not only cumbersome and time-consuming, but the relatively large number of steps required increases the risk of degradation, sample loss or cross-contamination of samples especially when several samples are processed simultaneously. In the case of RNA isolation, the risk of DNA contamination is comparatively high.

Apart from laborious and time-consuming traditional methods, alternative separation techniques have been developed. Sorption processes based on (a) hydrogen-binding interaction with an underivatised hydrophilic matrix, typically silica, under chaotropic conditions, (b) ionic exchange under aqueous conditions by means of an anion exchanger, (c) affinity and (d) size exclusion mechanisms were used for DNA purification. Solid-phase systems which adsorb DNAsilica-based particles (Vogelstein and Gillespie 1979; Boom et al. 1990, 1999; Melzak et al. 1996; Tian et al. 2000; Breadmore et al. 2003), glass fibres, and anion-exchange carriers (Ferreira et al. 2000; Endres et al. 2003; Teeters et al. 2003)are used in chromatographic separation columns [e.g. DE 41 43 639 C2 (Qiagen GmbH)] for example.

These carriers are applied for DNA isolation or purification together with highly concentrated chaotropic salt solutions (e.g. sodium iodide, sodium perchlorate, guanidinium thiocyanate). In US 5,075,430 (BioRad), for instance, usage of diatomaceous earth as a carrier material is described. Again, bonding takes place in the presence of a chaotropic salt. Other approaches are based on detergence together with a nucleic-acid-binding material (EP 0 796 327 B1, Dynal) or on the usage of a solid carrier with DNA-binding functional groups combined with polyethylene glycol and salts at high concentrations (WO/1999/058664, Whitehead Institute for Biomedical Research).

The increasing use of magnetic solid carriers in biochemical and molecular biology processes has many advantages compared to other non-magnetic separation processes. The term magnetic means that the support obtains a magnetic moment when placed in a magnetic field. Thus, it can be displaced. In other words, particles having a magnetic moment may be removed readily by the application of a magnetic field, e.g. by using a permanent magnet. This is a quick, simple and efficient way to separate the particles after the nucleic binding or elution step (see Fig.1) and a far less rigorous method than traditional techniques, such as centrifugation, that generate shear forces which may lead to the degradation of the nucleic acids. It is also possible to isolate components of the cell lysate, which inhibit for example the DNA polymerase of a following PCR reaction like polysaccharides, phenolic compounds or humic substances (Demeke and Adams 1992; Watson and Blackwell 2000).

Usually, it is sufficient to apply a magnet to the side of the vessel containing the sample mixture for aggregating the particles near the wall of the vessel and pouring away the remainder of the sample (see Fig.1).

Magnetic carriers with immobilised affinity ligands or prepared from a biopolymer exhibiting affinity to the target nucleic acid are used for the isolation process. Many magnetic carriers are commercially available and can also be prepared in the laboratory. Such materials are magnetic particles produced from different synthetic polymers, biopolymers, porous glass, or magnetic particles based on inorganic magnetic materials such as surface-modified iron oxide. Especially suited are superparamagnetic particles, which do not interact among each other in the absence of a magnetic field. These particles will magnetise under a strong magnetic field, but retain no permanent magnetism once the field is removed. When magnetic aggregation and clumping of the particles are prevented during the reaction, easy suspension of the particles and uniform nucleic acid extraction are ensured.

The diameter of the particles is approximately between 0.5 and 10m. Materials with a large surface area are preferred for binding the nucleic acids. Without going into theoretical details, the nucleic-acid-binding process may be assisted by the nucleic acid wrapping around the support. Such supports generally have an irregular surface and may be porous for example. Particulate materials, e.g. beads and in particular polymer beads, are generally preferred due to their larger binding capacity. Conveniently, a particulate solid support used will comprise spherical beads.

In the laboratory, colloidal magnetite Fe3O4 (or similar magnetic material such as maghemite Fe2O3 or ferrites) particles usually are surface-modified by silanisation. Naked iron oxide (Fe3O4) has the capacity of adsorbing DNA (Davies et al. 1998), but aggregates due to attractive forces reduce the surface area that can be used for adsorption. Silane compounds coupled to magnetite derivatised with carboxyl groups are known to have a DNA extraction ability in solutions containing PEG (Hawkins et al. 1994). Modified bacterial magnetite particles in the presence of amino silane compounds and hyperbranched polyamidoamine dendrimer are used for DNA extraction by Yoza et al. (2002, 2003). Modified magnetic cobalt ferrite particles have been investigated for DNA isolation under high sodium chloride and PEG concentrations by Prodelalova et al. (2004).

Surface modification of magnetic nanoparticles with alkoxysilanes (Bruce et al. 2004; Tan et al. 2004; Bruce and Sen 2005) or polyethyleneimine (Chiang et al. 2005; Veyret et al. 2005) is also useful. The above-mentioned magnetic colloids are not easy to separate using classical magnets. This is due to a small particle size, at which Brownian motion forces are higher than the exerted magnetic force. To enhance phase separation, various magnetic latexes that may interact with nucleic acids were prepared.

Magnetic micro-beads can be prepared in a number of ways, but usually magnetically susceptible particles (e.g. iron oxide) are coated with synthetic or biological polymers. Elaissari et al. (2003) describe the interaction of nucleic acids and different polymers. Biopolymers such as agarose, chitosan, -carrageenan, and alginate, can be prepared easily in a magnetic form (Levison et al. 1998; Prodelalova et al. 2004). In the simplest case, the biopolymer solution is mixed with magnetic particles and, after bulk gel formation, the magnetic gel formed is broken into fine particles. Alternatively, the biopolymer solution containing dispersed magnetite is dropped into a mixed hardening solution or a water-in-oil suspension technique is used to prepare spherical particles. Basically, the same process can be used to prepare magnetic particles for nucleic separation from synthetic polymers such as hydrophobic polystyrene (Ugelstad et al. 1992) and hydrophilic polyacryl amide (Elaissari et al. 2001) or poly(vinyl alcohol) (Oster et al. 2001). Genomic DNA was also successfully isolated from cell lysate on weak acid derivatives of magnetic P(HEMA-co-EDMA) and P(HEMA-co-GMA) microparticles in the presence of PEG and sodium chloride (Horak et al. 2005).

The first approach to synthesising micro-sized particles was published by Ugelstad et al. They developed an interesting methodology leading to monosized polystyrene magnetic microspheres, which were studied in various biomedical applications (Ugelstad et al. 1993). These particles have an excellent size distribution and spherical shape, but their surface is very hydrophobic and results in a high amount of unspecific protein binding on the particle surface.

Another possibility consists in combining different polymer matrix materials with silica components (Grttner et al. 2001; Mller-Schulte et al. 2005) that specifically interact with the nucleic acids.

Depending on the support and the nature of the subsequent processing required, it may or may not be desirable to release the nucleic acid from the support. The direct use of magnetic beads, e.g. in PCR or other amplifications, without eluting the nucleic acid from the surface is not trivial. The enzymatic detection and amplification methods will be inhibited by the magnetic beads, their stabilisers, or their metal oxides (Spanova et al. 2004), which decrease PCR sensitivity or lead to false negative PCR results. For many DNA detections or identification methods, elution is not necessary. Although the DNA may be randomly in contact with the bead surface and bound at a number of points by hydrogen binding or ionic or other forces, there generally will be sufficient lengths of DNA available for hybridisation to oligonucleotides and for amplification. If desired, however, elution of the nucleic acid may be achieved using known methods, e.g. higher ionic strength, heating or pH changes.

Commercially available magnetic particles that are suited for nucleic acid separation can be obtained from a variety of companies. Mostly, the matrixes are based on silica, porous glass, cellulose, agarose, polystyrene and silane (see Tables1 and 2). Moreover, some important patents exist that describe the synthesis of magnetic carriers not only for nucleic separation:

One of the first patents for particle synthesis is the Ugelstad polymerization process, which is described, for example, in EP 0 003 905 B2, US 5,459,378, and US 4,530,956 (SINTEF). It leads to monodisperse magnetic particles by several swelling and polymerisation steps. WO/1992/016581 (Cornell Research Foundation) also describes the preparation of monodisperse particles, particularly macroporous polymer beads. The process proposed uses a three-phase emulsion containing soluble polymer particles, a monomer phase and water. Nucleic acid separation using magnetic beads is described in (Alderton et al. 1992) and in WO/1991/012079 as well as in US 5,523,231 (Amersham). These magnetic beads are able to absorb the nucleic acid after a salt-ethanol precipitation. The approaches are not nucleic-acid-specific, i.e. the magnetic beads adsorb other bio-substances in parallel. Of course, this is a drawback of these approaches.

In the declaration WO/1996/041811 (Boehringer; Roche) mainly non-porous glass particles comprising mica and magnetite particles are described (Bartl et al. 1998). During their production, magnetic particles and a surrounding glass coating are superimposed on a mica core. The disadvantage of these products is their affinity to sedimentation. Furthermore, the production process is time-consuming and based on a complex spray process. Another approach to the production of particles from spherical magnetite kernels with a surface coating of silicon dioxide is covered by the European patent application EP 1 468 430 A1.

Monodisperse magnetic beads are described in WO/1998/012717 (Merck). They consist of a SiO2 core, which is given magnetic properties by a ferric-oxide coating. After a subsequent silanisation of the ferric-oxide coating, the particles can bind nucleic acids.

Many patents concerning nucleic acid separation are from the Dynal company. They developed monodisperse polymer magnetic particles with different sizes (coefficient of variation less than 5%) (see EP 0 796 327 B1), which are sold with a polystyrene matrix under the name of Dynabeads. The small-size distribution ensures reproducible separation properties. Protocols for nucleic acid separation with these particles are described by EP 0 512 439 B1 and with oligonucleotide-linked particles for specific nucleic acid separation in US 5,512,439.

Magnetic beads based on mica or polystyrene and coated by a magnetic oxide reach a high specific density, which leads to a fast sedimentation. Thus, additional mechanical mixing is necessary. The main drawback of the coated particles consists in the fact that the metal oxides may be in direct contact with the analytical solutions despite silanisation. All state-of-the-art approaches to the production of magnetic beads are laborious; the production process time amounts to several hours. To overcome this problem, the US patents 6,204,033 and 6,514,688 (chemagen Biopolymer Technologie AG) describe spherical, magnetic polymer particles based on polyvinyl alcohol particles, which can be produced in short terms using inverse suspension polymerisation. The polymer particles contain reactive hydroxyl groups to which other molecules can be coupled. Due to their hydrophilic surface, the particles exhibit small unspecific bindings only. Together with an at least partly silanised surface (DE 100 13 955 A1 and EP 1 274 745 A1) or a germanium-containing compound (DE 101 03 652 A1), they can be used for specific nucleic acid separation.

The inverse suspension process for the separation of nano- and micro-sized silica particles is suggested in WO/2002/009125 (Fraunhofer-Gesellschaft). The main idea is the dispersion of aqueous silica-sole containing magnetic colloids, which are hardened to spherical hydrophilic gel particles by adding a suited base. These particles can be used for nucleic acid separation with high binding capacities (WO/2005/50 52 581 A3, MagnaMedics GmbH).

Both total DNA and RNA are separated by the same magnetic beads. For the purpose of removing RNA from DNA, the RNA is destroyed before the DNA separation step. Adding of an RNAse or an alkali such as NaOH is an appropriate process. Vice versa, RNA can be separated if the DNA is degraded with DNAse.

The primary method considered for plasmid purification is the separation of plasmid DNA (pDNA) from the chromosomal DNA and cellular RNA of the host bacteria. Stadler et al. (2004) show that even in the case of a high copy plasmid, pDNA represents not more than 3% of the cleared lysate and that most of the critical contaminants are negatively charged (RNA, cDNA, endotoxin) and similar in size (cDNA, endotoxins) and hydrophobicity (endotoxins). A number of methods have been developed to generate a cleared lysate, but they are not able to remove proteins and lipids. Alkaline lysis of harvested bacterial cells with a subsequent neutralisation, as originally described by Birnboim and Doly (1979), is the process of choice. Cleared lysate protocols may vary slightly from each other as regards salt concentrations, volume, pH, temperature, and process step durations (Hirt 1967; Holmes and Quigley 1981; Birnboim 1983). These techniques make use of the differences in denaturation and renaturation characteristics of covalently closed circular plasmid DNA and chromosomal DNA fragments.

Table1 shows some commercially available magnetic particles used for DNA, RNA and pDNA isolation. Many magnetic particles are available with optimised buffers and protocols for small lab scale and automated systems. There are also some companies offering particles for nucleic acid purification without any further information.

The magnetic carrier is provided with binding solutions to assist in the selective capture of nucleic acids. For example, complementary DNA or RNA sequences (Satokari et al. 2005) or DNA-binding proteins may be used as well as viral proteins binding to viral nucleic acids. In this review, a short overview of eukaryotic mRNA and viral DNA/RNA will be given.

There are several companies (see Table2) offering oligodeoxythymidine immobilised with magnetic particles, which can be used effectively for the rapid isolation of highly purified mRNA from eukaryotic cell cultures or total RNA preparations (Jacobsen et al. 2004). These procedures are based on the hybridisation of the oligonucleotide dT sequence with the stable polyadenylated 3 termini of the eukaryotic mRNA. The length of the complementary sequence differs between 20 and 30 oligonucleotides. This sequence is directly bound covalently to the particle surface or indirectly by biotinylated oligonucleotides and the interaction of streptavidin-coated particles. CPG and Dynal (now Invitrogen) offer MPG and Dynabeads with already immobilised biotinylated oligonucleotide, but also other companies offer streptavidin-modified particles, which can be used for mRNA isolation, as described, e.g. by the mRNA isolation kit with MagneSphere from Promega. Nearly all magnetic particles (except for MagaCell oligo-dT30 and Sera-Mag oligo-(dT)30) are available together with an optimised buffer system and helpful protocols.

Automated extraction of viral RNA and DNA from the plasma mini-pool is performed by the chemagic Viral DNA/RNA Kit and chemagic Magnetic Separation Module I (Hourfar et al. 2005a,b; Pichl et al. 2005).

A rapid diagnosis of enterovirus infection by magnetic bead extraction has been established by Muir et al. (1993). Enterovirus RNA can be separated from large-volume water samples using the NucliSens miniMAG System (Rutjes et al. 2005). Hei and Cai (2005) developed a system for purifying SARS coronavirus RNA by a hybridisation of a specific oligonucleotide sequence, which is immobilised on the magnetic bead surface.

A variety of magnetic separators are available on the market, ranging from very simple concentrators for one tube to complicated fully automated devices. The isolation of nucleic acids is mostly performed in the batch mode using commercially available lab-scale magnetic separators (particle concentrators). Separators are usually made of strong rare-earth permanent magnets designed to hold various amounts of micro-tubes or tubes.

Particles with a diameter larger than 1m can be separated easily using simple magnetic separators, while separation of smaller particles (magnetic colloids with a particle size ranging from ten to hundreds of nanometres) may require the use of high-gradient magnetic separators.

The racks are designed to hold various amounts of micro-tubes or tubes. Test tube magnetic separators allow to separate magnetic particles from volumes between approximately 5l and 50ml. There are many combinations with other features like a mixing function (Ademtech) or a possibility to turn the separator over for the removal of the supernatant (chemagen Biopolymer-Technologie AG). Other devices are applied for the separation of magnetic particles from the wells of standard micro-titration plates. In some of them the temperature can be pc-controlled (AGOWA), other devices may be inserted into automated separation devices.

Laboratory automation is increasingly important in molecular biology and biotechnology. Constantly increasing numbers of analyses of different sources and sample volumes have resulted in an enormous importance of flexible robots or automated systems. Automation is also required for handling a large number of samples without human errors.

Many instruments have been developed to automate PCR amplification, the sequencing reaction and the detection of nucleic acids, but automating DNA extraction by traditional methods with centrifugation and vacuum steps still is difficult. A complete separation of the solid carrier matrix by centrifugation is not possible. Supports filled with carrier materials cannot be used, as the ineluctable dead volumes of the support lead to sample material loss in case of small amounts of sample materials. Another drawback is the danger of mutual contamination of different biological samples, especially if directly neighbouring supports are emptied by the vacuum. However, the last decade shows that DNA purification using magnetic bead technology is suitable for automation systems, and several automated instruments for handling magnetic beads have been developed (Alderton et al. 1992; Wahlberg et al. 1992; Rolfs and Weber 1994; Fangan et al. 1999; Obata et al. 2001; Akutsu et al. 2004; Vuosku et al. 2004).

More and more vendors offer commercially automated devices for the handling of magnetic particles, e.g. for the purification of nucleic acid (see Table4). Most systems are offered together with system-specific optimised particles, buffer systems and protocols.

The devices are able to process between six and 96 samples in parallel and commonly customised for small buffer volumes. For larger volumes, the chemagic Magnetic Separation Module I (<10ml) (see Fig.2) or the Magtration System 8l(7ml) can be used.

chemagic Magnetic Separation Module I consisting of (A) separation head with magnetizable rods [here 12-well format for large (50ml) volumes; 96-well format for MTPs also available], (B) electro magnet, (C) chemagic dispenser for parallel filling of all required buffer solutions (accessory) and (D) tracking unit. The principle functionality regarding separation and resuspension of magnetic beads is shown in the scheme

The present review has shown that the separation of nucleic acid is a highly dynamic field of research and development. An increasing number of commercial vendors offer magnetic particles, also in the form of a kit that is optimally suited for the application desired. The increasing number of publications shows that magnetic particles of higher potential are currently under research. Materials with more specific-binding properties and a better separability are promising approaches. A higher degree of automation leads to systems analysing a larger number of samples and higher sample volumes at the same time.

Akutsu J-I, Tojo Y, Okochi M, Yohda M, Segawa O, Obata K, Tajima H (2004) Development of an integrated automation system with a magnetic bead-mediated nucleic acid purification device for genetic analysis and gene manipulation. Biotechnol Bioeng 86:667671

Boom R, Sol C, Beld M, Weel J, Goudsmit J, Wertheim-van Dillen P (1999) Improved silica-guanidinium thiocyanate DNA isolation procedure based on selective binding of bovine alpha-casein to silica particles. J Clin Microbiol 37:615619

Breadmore MC, Wolfe KA, Arcibal IG, Leung WK, Dickson D, Giordano BC, Power ME, Ferrance JP, Feldman SH, Norris PM, Landers JP (2003) Microchip-based purification of DNA from biological samples. Anal Chem 75:18801886

Fangan BM, Dahlberg OJ, Deggerdal AH, Bosnes M, Larsen F (1999) Automated system for purification of dye-terminator sequencing products eliminates up-stream purification of templates. Biotechniques 26:980983

Hourfar MK, Schmidt M, Seifried E, Roth WK (2005) Evaluation of an automated high-volume extraction method for viral nucleic acids in comparison to a manual procedure with preceding enrichment. Vox Sang 89:7176

Jacobsen N, Nielsen PS, Jeffares DC, Eriksen J, Ohlsson H, Arctander P, Kauppinen S (2004) Direct isolation of poly(A)(+) RNA from 4M guanidine thiocyanate-lysed cell extracts using locked nucleic acid-oligo(T) capture. Nucleic Acids Res 32:e64

Muir P, Nicholson F, Jhetman M, Neogi S, Banatvala JE (1993) Rapid diagnosis of enterovirus infection by magnetic bead extraction and polymerase chain-reaction detection of enterovirus RNA in clinical specimes. J Clin Microbiol 31:3138

Obata K, Segawa O, Yakabe M, Ishida Y, Kuroita T, Ikeda K, Kawakami B, Kawamura Y, Yohda M, Matsunaga T, Tajima H (2001) Development of a novel method for operating magnetic particles, Magtration Technology, and its use for automating nucleic acid purification. J Biosci Bioeng 91:500503

Rutjes SA, Italiaander R, van den Berg HHJL, Lodder WJ, de Roda Husman AM (2005) Isolation and detection of enterovirus RNA from large-volume water samples by using the nucliSens miniMAG System and real-time nucleic acid sequence-based amplification. Appl Environ Microbiol 71:37343740

Vuosku J, Jaakola L, Jokipii S, Karppinen K, Kamarainen T, Pelkonen VP, Jokela A, Sarjala T, Hohtola A, Haggman H (2004) Does extraction of DNA and RNA by magnetic fishing work for diverse plant species? Mol Biotechnol 27:209215

DE 101 03 652 A1 Magnetische Polyvinylalkoholpartikel mit modifizierter Oberflche zur Isolierung und Reinigung von Nukleinsuren (2002) Brassard L, Parker J, Smets H, Oster J; chemagen Biopolymer-Technologie AG, Germany

US 4,336,173 Process for preparing an aqueous emulsion or dispersion of a partly water-soluble material, and optionally further conversion of the prepared dispersion or emulsion to a polymer dispersion when the partly water-soluble material is a polymerizable monomer (1980) Ugelstad J; SINTEF, Norway

US 4,530,956 Process for the preparation of aqueous dispersions of organic materials and possible further conversion to a polymer dispersion when the organic material is a polymerizable monomer (1985) Ugelstad J, Berge A; SINTEF Norway

WO/2002/009125 Spherical, magnetic SiO2 particles with an adjustable particle and pore size and an adjustable magnetic content. Method for producing them and use of SiO2 particles of this type (2001) Mller-Schulte D, Fischer R; Fraunhofer-Gesellschaft zur Frderung der angewandten Forschung e.V. Germany

fractionated mitochondrial magnetic separation for isolation of synaptic mitochondria from brain tissue | scientific reports

fractionated mitochondrial magnetic separation for isolation of synaptic mitochondria from brain tissue | scientific reports

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While mitochondria maintain essential cellular functions, such as energy production, calcium homeostasis, and regulating programmed cellular death, they also play a major role in pathophysiology of many neurological disorders. Furthermore, several neurodegenerative diseases are closely linked with synaptic damage and synaptic mitochondrial dysfunction. Unfortunately, the ability to assess mitochondrial dysfunction and the efficacy of mitochondrial-targeted therapies in experimental models of neurodegenerative disease and CNS injury is limited by current mitochondrial isolation techniques. Density gradient ultracentrifugation (UC) is currently the only technique that can separate synaptic and non-synaptic mitochondrial sub-populations, though small brain regions cannot be assayed due to low mitochondrial yield. To address this limitation, we used fractionated mitochondrial magnetic separation (FMMS), employing magnetic anti-Tom22 antibodies, to develop a novel strategy for isolation of functional synaptic and non-synaptic mitochondria from mouse cortex and hippocampus without the usage of UC. We compared the yield and functionality of mitochondria derived using FMMS to those derived by UC. FMMS produced 3x more synaptic mitochondrial protein yield compared to UC from the same amount of tissue, a mouse hippocampus. FMMS also has increased sensitivity, compared to UC separation, to measure decreased mitochondrial respiration, demonstrated in a paradigm of mild closed head injury. Taken together, FMMS enables improved brain-derived mitochondrial yield for mitochondrial assessments and better detection of mitochondrial impairment in CNS injury and neurodegenerative disease.

Mitochondria are small (0.5 to 2 m) organelles that provide a majority of the cells energy in the form of adenosine triphosphate (ATP). In addition to providing the cell with ATP, mitochondria are responsible for regulating calcium homeostasis, cell signaling via metabolic shifts and apoptotic pathways. Furthermore, mitochondrial function is particularly important in the brain as the central nervous system (CNS) consumes around 20 percent of the bodys oxygen1. Specifically, mitochondria located in the neural synapse may undergo the highest bioenergetic demand in the brain as they supply energy needed during neurotransmission2. Synaptic mitochondria are highly involved in the regulation of neurotransmitter release3 and synaptic vesicle formation4. Other mitochondria in the brain (non-synaptic) can be derived from multiple cell types or locations, such as astrocytes, microglia, endothelial cells, and even neuronal soma. These perform essential roles, such as modification of microRNA activity5, and produce energy for various cellular activities.

The role of mitochondria in human disease continues to expand as new studies highlight their impact in a variety of medical disciplines6. Mitochondrial dysfunction is well established as a key pathological mechanism in neurodegenerative disease7,8. Moreover, synaptic mitochondria have been shown to be more vulnerable to calcium overload, aging and neurotrauma9,10,11,12, likely due to higher basal energy demand and pathological damage to the synapse. In neurodegenerative disorders8,13,14, significant changes in mitochondrial fission and fusion occur, contributing to variation in mitochondrial density at the synapse. Due to this specific susceptibility in neurodegeneration, it is crucial to obtain specific mitochondrial sub-populations, synaptic and non-synaptic fractions, to pinpoint functional changes. For functional measurements, synaptic mitochondria are extracted from synaptoneurosomes, which are formed during brain tissue homogenization.

Isolation techniques have been developed to keep mitochondria intact and functional, preserving their physical and biochemical characteristics. Two centrifugation methods for isolating mitochondria from CNS tissue are differential centrifugation (DC) and density gradient high speed/ultracentrifugation (UC). While these methods each have specific advantages that render them useful in different scenarios (Table1), they have limitations for assessment of highly susceptible synaptic mitochondria, detailed below. The quickest and most inexpensive method for isolating mitochondria is DC, which utilizes centrifugation, ranging from 1000g to 15,000g, to isolate mitochondria from other cellular organelles (Fig.1). DC yields total mitochondria, but also captures cellular debris that may interfere with downstream assays assessing mitochondrial activity15. This inability to isolate high-purity mitochondrial pelletsor specific mitochondrial sub-populations creates the need for more intricate, albeit more expensive and time-consuming, techniques of mitochondrial isolation.

Schematic of mitochondrial isolation techniques. (Top) Workflow of differential centrifugation (DC) to isolate total mitochondria from mouse brain samples. (Middle) Workflow of density gradient ultracentrifugation (UC) to isolate synaptic and non-synaptic mitochondrial fractions from mouse brain samples. (Bottom) Workflow of fractionated mitochondrial magnetic separation (FMMS) technique to isolate synaptic and non-synaptic mitochondrial fractions from mouse brain samples. Illustration by Matt Hazzard, University of Kentucky, Information Technology.

Purified mitochondrial fractions have been obtained previously using Percoll gradient high speed centrifugation16,17,18. This technique requires a Percoll gradient step (at least 18,000g spin16) to purify the non-synaptic, or free, mitochondrial fraction18. Additionally, this technique has been utilized to isolate non-synaptic mitochondria from small regions of the mouse brain, demonstrating relative high yield and purity16. While these reports using Percoll gradients do not include disruption of synaptoneurosomes and further isolation of synaptic mitochondria, our laboratory has used this technique to isolate both synaptic and non-synaptic mitochondrial populations9,19, though requiring higher speed centrifugation (30,400g spin). In addition, these Percollpreparations result in phase bands of mitochondria rather than distinct mitochondrial pellets.

UC is a more specific isolation technique that utilizes exceedingly high speed centrifugation and buoyancy to allow for separation of non-synaptic and synaptic mitochondria9,20,21. In order to separate non-synaptic mitochondria from synaptoneurosomes, a Ficoll sucrose density gradient is used and free mitochondria are pelleted, as opposed to phase bands obtained during Percoll preparations (Fig.1). While the UC approach allows for pure non-synaptic and synaptic mitochondria to be isolated, this method is expensive, requires technical proficiency and can be time-consuming when running many samples. A common issue with UC is being unable to obtain sufficient synaptic mitochondria yield for technical analysis from low initial tissue amounts, limiting its application to small brain regions such as the hippocampus in mice. In general, UC, similar to DC, requires many hands-on steps that can result in damage or loss of mitochondria (i.e. pipetting and supernatant transfer)22. Thus, there is a need for a mitochondrial isolation technique, such as affinity purification, that can preserve mitochondria yields and mitochondrial heterogeneity when using lower amounts of brain tissue.

The use of magnetically labeled antibodies for mitochondrial extraction has been shown to yield intact functional mitochondria from cell culture and various tissues15,23,24,25, but a protocol for isolating brain synaptic and non-synaptic mitochondria using this method has yet to be refined. Although the magnetic-activated cell sorting (MACS) system has been previously been utilized to purify synaptosomal mitochondria26, this procedure requires usage of density gradient UC. Magnetic mitochondrial isolation can be faster than UC techniques, depending on the specific application27,28.

In this study, we detail how to apply magnetic immunolabeling, using the MACS system, to target the Tom22 protein on the outer membrane of the mitochondria for synaptic and non-synaptic mitochondrial isolation. Fractionated mitochondrial magnetic separation (FMMS) optimization using magnetic labeling is described, including antibody titration to determine the optimal concentration for mitochondrial saturation. In addition, FMMS is compared to other mitochondrial isolation techniques to demonstrate its advantages (see Table1). The current study is the first to demonstrate how the MACS system can be solely utilized to isolate, purify, and separate brain-derived non-synaptic and synaptic mitochondria without the use of UC (Fig.1). This is widely relevant to any laboratory focused on assessing neurodegenerative disease or CNS injury without purchase of an ultracentrifuge. Finally, this protocol is applied to examine mitochondrial respiration in a paradigm of mild traumatic brain injury(TBI). Similar injury models demonstrate that mitochondrial fusion occurs after this injury, which increases mitochondrial heterogeneity14. We show that the newly developed FMMS technique can provide better resolution of mitochondrial function and higher mitochondrial yields from mouse brain tissue, eliminating the need to pool tissues from multiple animals.

One modification to the manufacturers protocol was to adjust the concentration of tissue homogenate to buffer for antibody incubation and magnetic column loading. In the manufacturers protocol, 10mL of buffer was recommended for 50 to 100mg of brain tissue. To provide an exact ratio of buffer to brain tissue homogenized and to optimize using our own mitochondrial isolation buffer, we titrated in this recommended range, as to not overload the magnetic column, and tested yield and mitochondrial respiration (data not shown). We found that a concentration of 10mL buffer per 75mg of tissue was optimal for both outcome measures, which is within their recommendation.

A previous study by Franko, et al. reported that labeling mouse brain tissue with 0.5L antibody per mg of tissue resulted in a lack of saturation of mitochondria by magnetic beads15. To build upon these data and ensure that all free mitochondria were labeled, we ran assays of differing antibody to tissue concentrations measuring protein output. The range of antibody concentrations (1 to 6L of antibody per 1mg of tissue) showed major differences in protein yield suggesting that the manufacturer recommended antibody concentration (0.51L/mg tissue) is not sufficient for mouse brain mitochondria saturation. In the cortex, we found that the antibody titration for non-synaptic mitochondria demonstrated a plateau effect, indicating saturation (Fig.2a), while a similar trend was shown for non-synaptic hippocampus-derived mitochondrial protein (Fig.2b). Based on these observations, it is recommended to use >3L/mg tissue for cortical non-synaptic mitochondria and >4L/mg tissue for hippocampal non-synaptic mitochondria, using the prescribed procedure in this manuscript.

Non-synaptic fraction optimization (a) Cortical brain homogenates were centrifuged at low speed (1300 g) and the supernatant was collected (procedure performed twice). This combined supernatant containing cortical non-synaptic mitochondria was incubated with 1, 1.5, 2, 3, and 4L antibody/mg tissue and total protein yield was calculated. (b) Hippocampal brain homogenates were centrifuged at low speed (1300g) and the supernatant was collected (procedure performed twice). This combined supernatant containing hippocampal non-synaptic mitochondria was incubated with 1, 3, 4 and 6L antibody/mg tissue and total protein yield was calculated. (c) Non-synaptic mitochondrial fractions were obtained from mouse hippocampus using the UC protocol and FMMS protocol. Mitochondrial yield (g) was normalized to initial brain tissue amount (mg). (d) Non-synaptic mitochondrial fractions were obtained using the UC protocol and FMMS protocol. The Seahorse XFe24 Flux Analyzer was utilized to measure oxygen consumption rates (OCR) from these samples. Respiratory control ratio (RCR) was calculated by dividing State III OCR respiration values by State IV OCR respiration values. No significant difference was observed. N=36/group. Bars+error bars correspond to MeanSEM.

For the FMMS technique, it is imperative to saturate the non-synaptic mitochondria with antibody to prevent free, unbound non-synaptic mitochondria from passing into the eluate during the first pass through the magnet (Fig.1). This would lead to non-synaptic mitochondria in the synaptosomal fraction, biasing the mitochondrial population and leading to potentially inaccurate results. Thus, we increased the non-synaptic antibody concentration in our final protocol to 4L of magnetic antibody solution per mg of initial brain tissue to ensure total saturation. Increasing the antibody concentration for the non-synaptic pulldown also ultimately lead to a ~1.5x increase in mitochondrial yield compared to UC methods (Fig.2c). Additionally, RCRs were comparable, even slightly higher, in mitochondria obtained from FMMS compared to those obtained by UC (Fig.2d).

After determining the appropriate antibody concentration for non-synaptic mitochondria, we optimized antibody concentration for the synaptic fraction. After collecting the wash-through from the first magnet pass, synaptoneurosomes were pelleted by centrifugation. Afterwards, nitrogen disruption of synaptoneurosomes was completed to release synaptic mitochondria29. Due to the relatively smaller amount of synaptic mitochondria, a lower antibody concentration range was tested (0.2 to 1L of antibody per mg of tissue). As with the non-synaptic fraction, the yield was dependent on antibody concentration. A change in synaptic antibody concentration from 0.2 to 1L per mg of tissue resulted in a 5x increase in yield in the hippocampus and an even greater increase in yield in the cortex (Fig.3a). Furthermore, FMMS yielded 3x more synaptic mitochondria compared to UC (Fig.3b). Corroborating previous reports, RCR values of synaptic mitochondrial preparations were similar between FMMS and UC, although FMMS-derived samples demonstrated higher values (Fig.3c)28. Thus, the FMMS protocol isolates intact and functional synaptic mitochondria from the mouse brain.

Synaptic fraction optimization. (a) Hippocampal and cortical synaptic mitochondrial fraction were separately saturated with 4L antibody/mg tissue and pulled through the magnetic column. The resulting eluate containing synaptoneurosomes was nitrogen bombed to release synaptic mitochondria. The supernatant containing hippocampal or cortical synaptic mitochondria was incubated with 0.2 or 1L antibody/mg tissue and total protein yield was calculated. (b) Synaptic mitochondrial fractions were obtained from mouse hippocampus using the UC protocol and FMMS protocol. Mitochondrial yield (g) was normalized to initial brain tissue amount (mg). FMMS technique produced significantly higher (p<0.01) levels of mitochondrial protein compared to UC methods. (c) Synaptic mitochondrial fractions were obtained using the UC protocol and FMMS protocol. The Seahorse XFe24 Flux Analyzer was utilized to measure oxygen consumption rates (OCR) from these samples. Respiratory control ratio (RCR) was calculated by dividing State III OCR respiration values by State IV OCR respiration values. No significant difference was observed. N=36/group. Bars+error bars correspond to MeanSEM.

Previous studies have shown that DC-derived mitochondrial samples contain cellular debris that can be eliminated with an additional magnetic separation step15,30. We evaluated our UC- and FMMS-derived synaptic fractions using three different markers, NDUAF9 (mitochondria), tubulin (cytosol), and calnexin (mitochondria-associated membrane). We found that, as expected, both NDUAF9 and calnexin levels were similar for the two isolation methods. However, tubulin levels were significantly lower (p<0.05) in the FMMS-derived sample, indicating a higher purity can be obtained compared to UC (Fig.4).

Markers of mitochondrial fraction purity after either UC or FMMS procedures. (Left) Protein levels, obtained by western blot, were normalized to UC values. While NDUAF9 and calnexin levels were not significantly different between the groups, tubulin levels were significantly lower after FMMS methods compared to UC methods. (Right) Representative western blot for all markers. These blots were cropped from different parts of the same gel for clarity. Full-lengths blots are provided in the Supplementary material (Fig.S1). N=57/group. *p<0.05 compared to UC. Bars+error bars correspond to MeanSEM.

In applying this protocol, we used a model of repeated mild TBI that has previously shown mitochondrial deficits31. Building upon data demonstrating a State III respiration decrease after injury (t=2.843; p=0.0294) in total (DC) mitochondria31, we employed UC and FMMS protocols to assess synaptic and non-synaptic fractions. We expect that UC or FMMS can resolve which mitochondrial fraction displays the highest level of dysfunction. A disadvantage of the UC method is that it required both hippocampi from three mice be pooled in order to achieve enough synaptic mitochondria protein to run the respiration assay in triplicate. In bilateral hippocampus (40mg wet tissue), we did not observe any significant changes in State III respiration from either synaptic or non-synaptic mitochondrial fractions using the UC isolation procedure. In contrast, FMMS could be performed on hippocampal tissue (35mg wet weight) from individual mice exposed to the same injury paradigm. Analysis of the FMMS-derived non-synaptic mitochondrial fraction revealed a statistically significant decrease (t=2.521; p=0.0284) in State III function compared to the sham group (Fig.5). This suggests that FMMS, in addition to increasing yield, produces a higher level of resolution, potentially by capturing a higher percentage of damaged mitochondria that would be lost during the UC procedure.

Application of FMMS in a model of repeated closed head injury. Mice were given either a repeated CHI (rCHI) at a 48h interval or sham procedure. At 48h after the final CHI, bilateral hippocampus was extracted and homogenized for mitochondrial respiration assessment. (Left) Total mitochondria obtained through DC methods demonstrated a State III OCR decrease in the repeated CHI group compared to Sham. This data was modified from previously published work31. (Middle) Non-synaptic and synaptic fractions were obtained by UC procedures. Neither fraction showed any significant differences between repeated CHI and Sham groups. (Right) Non-synaptic and synaptic fractions were obtained by FMMS. While the synaptic fraction did not show any significant differences between repeated CHI and Sham groups, State III OCR was lower in the non-synaptic fraction of the repeated CHI group compared to Sham. N=6/group. *p<0.05 compared to Sham. Bars+error bars correspond to MeanSEM.

The current standard method for isolating synaptic and non-synaptic mitochondrial fractions is UC with the use of a sucrose density gradient. While UC effectively separates these fractions in large brain tissue samples, there are limitations in obtaining sufficient synaptic mitochondrial yield from small amounts (<60mg) of brain tissues. In addition to the lack of mitochondrial yield, there is concern that UC preparations result in the loss of dysfunctional mitochondria thereby making it less sensitive for detecting changes associated with brain pathology. In contrast, FMMS can separate mitochondrial fractions in a relatively quick and effective manner, resulting in a higher yield of both functional and, in the context of neurological disease or injury, dysfunctional mitochondria with increased purity. Importantly, FMMS allows isolation of synaptic and non-synaptic mitochondria from small brain regions, such as mouse hippocampi, and provides increased sensitivity for detection of mitochondrial dysfunction relative to UC.

In this study, we optimized the use of the MACS system with magnetic anti-Tom22 antibodies for isolation of synaptic and non-synaptic mitochondria from the mouse hippocampus and cortex. Secondly, we modified the manufacturers protocol for extracting tissue amounts lower than the recommended range of 50100mg tissue. The optimal antibody to tissue concentration was determined by measuring non-synaptic mitochondrial protein yield until the saturation point was reached (Fig.2). Additionally, it was crucial to reach non-synaptic fraction saturation to prevent non-synaptic mitochondrial crossover into the synaptic fraction. While the non-synaptic cortical fraction had a saturation point around 3L antibody per mg of tissue, the hippocampal non-synaptic fraction saturated at 4L antibody per mg of tissue (Fig.2b). Hippocampal tissue could require a higher antibody concentration to saturate non-synaptic mitochondria either due to a higher cell density or higher mitochondria number per cell as compared to the cortex. Given these observations, specific brain regions should be independently optimized for antibody concentration. Antibody concentrations used for the synaptic mitochondrial fraction (Fig.3a) can be adjusted to achieve sufficient mitochondria needed for downstream assays.

One limitation of UC methods is the inability to sustain initial mitochondrial protein levels due to loss of mitochondria during multiple pipetting steps32 and density gradient fractionation. In pathological settings such as neurodegenerative disease, traumatic injury, or ischemic insult, mitochondria can be damaged or undergo fusion or fission13,14,33 resulting in a change in density that could preclude their capture during UC. This issue is mitigated by FMMS as, at mitochondrial saturation, every mitochondrion that expresses Tom22, a component of the translocase of the outer mitochondrial membrane, is theoretically labeled. Of course, ruptured mitochondria without an outer membrane would not be captured using this technique. All samples in this report include mitochondria that are bound to these magnetic microbeads. Due to the small size (50nm), biodegradability, and non-toxic nature of these microbeads (Miltenyi Biotec), they are compatible with all downstream applications and do not need to be removed which is an added advantage of FMMS. We corroborate this by showing comparable RCR values of FMMS-derived mitochondria compared to UC-derived mitochondria (Figs2 and 3)15.

We show that mitochondrial protein levels greatly increase when employing FMMS compared to UC (Fig.3b). Non-synaptic mitochondrial yield is similar to a previously published report on a Percoll gradient technique isolating free mitochondria from mouse striatum16, though this did not include subsequent synaptic mitochondria isolation. Previous reports detailing synaptic mitochondrial respiration within subregions of the mouse brain require pooling of tissues from multiple mice to create one sample34,35,36. A previous study has shown that the amount of mitochondrial protein was ~4x more using the MACS system compared to UC methods28, which is corroborated in the current study (Fig.3b). Our current technique would eliminate the need for animal pooling due to the much higher (3x more) mitochondrial protein yields in small brain regions compared to UC methods. In addition to animal reduction, this also would reduce variability inherent with pooled tissues while increasing experimental/statistical power by allowing for animal-to-animal correlations with other outcomes, such as behavioral tests. FMMS showed a 45% increase in non-synaptic yield and a 200% increase in synaptic yield compared to UC in hippocampal tissue (Figs2c and 3b). This increase in yield highlights the utility of our FMMS protocol for examining mitochondrial function in small brain regions.

Mitochondrial preparation using UC has the potential for selective loss of dysfunctional mitochondria, which are critical for evaluation of brain pathology. This decreases the mitochondrial heterogeneity, leading to an incomplete view of mitochondrial function (or dysfunction). As an example, mitochondrial dysfunction, initiated by repeated mild TBI31, was detected in a total mitochondria DC preparation as a decrease in State III respiration, but was not apparent after synaptic/non-synaptic fractionation by UC (Fig.5). However, when using FMMS, significant State III respiration decrease was again noted and was identified to preferentially involve the non-synaptic fraction, suggesting that damaged mitochondria are preserved using this method. Therefore, FMMS provides increased sensitivity to pinpoint regional and temporal mitochondrial changes in neurodegenerative disorders or after CNS injury. It is important to note that maintaining mitochondrial heterogeneity with FMMS would provide greater resolution in detecting therapeutic efficacy of mitochondrial-directed treatments in various neurological diseases.

The quality and purity of MACS-derived mitochondria, with respect to amounts of endoplasmic reticulum (ER) and nucleus contamination, has been shown to be comparable to the UC method28. A subsequent study reported less nuclear contamination with the MACS system than with DC and UC methods37. Additionally, combining DC with MACS separation achieves higher purity (less cytosol, ER, and nuclear contaminates) compared to either DC or MACS mitochondrial isolation alone30. Consistent with this study, we show that FMMS results in significantly lower amounts of cytosolic tubulin compared to UC methods (Fig.4). Overall, FMMS-derived mitochondrial samples have higher purity, with less cytosolic and potentially less ER contamination, compared to UC-derived mitochondrial samples. While MACS separation is reliable for respiration assays, it has limitations with other sensitive assays such as lipidomics due to some amounts of ER and nuclear fraction contamination38. An additional limitation is that the MACS kits and antibodies are expensive, especially compared to DC methods, although much less than the cost of an ultracentrifuge. Furthermore, antibody titration is required for each brain region utilized as it is crucial to reach non-synaptic fraction saturation to prevent non-synaptic mitochondrial crossover into the synaptic fraction.

The implications of this new and improved technique are far-reaching in the fields of CNS injury and neurodegenerative disease39. Using the current protocol, researchers will be able to harness the power of FMMS to assay small, but crucial, brain regions in individual mice providing a degree of regional specificity that was not previously possible. For example, FMMS could be utilized to assay hippocampal sub-regions important in aging12, Alzheimers Disease (AD)11 and neurotrauma40, or to assay mitochondria from the amygdala, a region associated with post-traumatic stress disorder (PTSD)41 and other stress-related neuropsychiatric disorders42. Basal ganglia in the mouse could potentially be examined now using FMMS, yielding new insights relevant to neurological disorders such as Parkinsons Disease (PD)43 and Huntingtons Disease (HD)44,45,46. Furthermore, using FMMS could reveal greater mitochondrial dysfunction than determined previously, based on higher sensitivity of the technique.

In this study, we identified the optimal procedures for isolating synaptic and non-synaptic mitochondria from the hippocampus and cortex using FMMS. The FMMS protocol presents several benefits over traditional UC isolation techniques. Advantages of this system (Table1) include the ability to derive synaptic and non-synaptic mitochondrial fractions from low amounts of neuronal tissue (35mg), while producing similar mitochondrial respiration profiles. Furthermore, FMMS yields significant increases in mitochondrial protein compared to UC techniques. Finally, using the magnetic system preserves the ability to measure mitochondrial respiration from a heterogeneous population of mitochondria, including damaged and undamaged mitochondria. This concept is highlighted when applying FMMS in a model of repeated mild TBI. This technique has wide-ranging implications in the field of brain metabolism and neurodegenerative disease. With FMMS, researchers can gaingreater understanding of how mitochondria within specific brain subregions are altered after pathological insult.

Meanwhile, prepare the density gradient in ultracentrifuge tubes by adding 2mL of 10% Ficoll solution followed by gently adding 2mL of 7.5% Ficoll solution (make sure a distinct interface can be seen between the two solutions in the tube).

Add the mitochondrial sample over top of the prepared Ficoll double layered solution and centrifuge at 100,000 rcf for 30minutes at 4C using ultracentrifuge to obtain synaptoneurosomes and non-synaptic mitochondria.

Critical Step: When collecting synaptosomal layer, take as little Ficoll solution as possible in the pipette. Transferring a large amount of Ficoll solution will result in an inability of synaptoneurosomes to pellet.

Add the mitochondrial sample over top of the prepared 10% Ficoll solution and centrifuge at 100,000 rcf for 30minutes at 4C using ultracentrifuge to purify synaptic mitochondria from other debris (the synaptic mitochondria will reside in the pellet).

Our protocol was adapted from the manufacturers mitochondrial isolation protocol, incorporating aspects of previously published work47. This isolation uses centrifugation steps followed by magnetic separation, similar to previous methodology30. Mouse brain tissues were rapidly dissected from nave C57BL/6J male mice (Jackson Laboratories, Bar Harbor, Maine). Samples were kept at 4C on ice and all reagents were previously stored at 4C to prevent degradation of the mitochondria. Mitochondria were isolated from brain tissue samples using mouse mitochondrial isolation kits (Miltenyi Biotec). A schematic of the procedure is shown in Fig.1.

Protein was quantified using a BCA analysis kit (Pierce, Cat # 23,227) and the absorbance was measured at 560nm on a Biotek Synergy HT plate reader (Winooski, Vermont). Anti-TOM22 magnetic microbeads present in our samples did not contribute to over-estimation of protein.

The mitochondrial bioenergetic measurements were carried out using a Seahorse XFe24 Extracellular Flux Analyzer (Agilent Technologies, USA), which determines the bioenergetics of mitochondria by measuring the Oxygen Consumption Rates (OCR) during various states of respiration. The OCR were measured in the presence of different substrates, inhibitors and un-couplers of the Electron Transport Chain (ETC) using previous methods with slight modifications31. The stocks used for the assays were 500mM pyruvate, 250mM malate, 30mM adenosine diphosphate (ADP), and 1M succinate (pH for all were adjusted to 7.2). Stocks of 1.26mM oligomycin A, 1mM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), 1mM rotenone were prepared in ethanol and used for assays. As per the instructions from the XFe24 Extracellular Flux kit, the sensor cartridge was hydrated overnight at 37C. The injection ports A to D of the sensor cartridge were then loaded with 75L of different combinations of the above substrates/inhibitors/uncouplers as follows. Before loading, the stocks were diluted appropriately in the respiration buffer (RB) to get the final concentrations in the respiration chamber of 5mM pyruvate/ 2.5mM malate/ 1mM ADP (via Port A), 1M oligomycin A (via Port B), 4M FCCP (via Port C) and 0.1M rotenone/10mM of succinate (via Port D) starting with the initial volume of 525L RB in the chamber and accounting for the addition of volume with every injection through ports A to D. Once loaded, the sensor cartridge was placed into the Seahorse XFe24 Flux Analyzer for automated calibration.

Seahorse Standard XFe24 assay plates were used for loading mitochondria. The MACS-purified non-synaptic and synaptic mitochondria were diluted to 5g and 10g, respectively, per 50L in RB and 50L was loaded in each well. The UC-purified non-synaptic and synaptic mitochondria were diluted to 2.5g and 5g, respectively, per 50L in RB and 50L of this solution was loaded in each well. The assay plates were centrifuged at 3,000rpm for 4min at 4C to adhere the mitochondria to the bottom of the wells. After centrifugation, 475L of RB (pre-incubated to 37C) was added without disturbing the mitochondrial layer to obtain a final volume of 525L per well. After the instrument calibration with the sensor cartridge was complete, the utility plate was replaced by the plate loaded with mitochondria for bioenergetics analysis. Briefly, it involved cyclic steps of mixing, sequential injections of substrates and inhibitors via Ports A thru D, mixing, equilibration, and measurement of the OCR and pH through fluorimetric optical probes. The data output consisted of State III respiration in the presence of pyruvate, malate and ADP (Port A) followed by State IV rate in presence of oligomycin A (Port B). Subsequently, uncoupled respiration State VPM (State V1) in the presence of FCCP (Port C) and State VSucc (State V2) in the presence of rotenone/ succinate (Port D) were assayed, respectively. Respiratory control ratio (RCR) was calculated by dividing State III respiration values by State IV respiration values.

All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Kentucky and complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Division of Laboratory Animal Resources at the University is accredited by the Association for the Assessment and Accreditation for Laboratory Animal Care, International (AAALAC, International) and all experiments were performed within its guidelines. All data were analyzed and reported according to ARRIVE guidelines. Young adult (79 week old; 2226g) C57BL/6J male mice (Jackson Laboratories, Bar Harbor, Maine) were acclimated for a one-week period to the vivarium where they were housed (five per cage) in a 14h/10h light/dark cycle with food and water available ad libitum. Mice were then randomly assigned to two groups: repeated closed head injury (CHI) at a 48h interval and sham. Animals (n=6/group) were euthanized before mitochondrial isolation at 48h after the final sham injury or CHI. For all assays, technical triplicates were included for each sample. Data analysis was performed blinded to treatment groups.

Experimental CHI was induced following a previously described procedure48. Mice were anesthetized with 2.5% isofluorane delivered via a nose cone and the head of each mouse was fixed between two zygomatic cuffs stabilized in a stereotaxic frame. A pneumatically controlled cortical impact device (TBI-0310 Impactor, Precision Systems and Instrumentation, Fairfax Station, VA) with a 5mm diameter, cushioned tip was programmed to deliver a 2.0mm impact at 3.5m/s with a 500ms dwell time. The posterior edge of the tip was aligned at the Lambda suture (approximately Bregma level 5mm). The diameter of the tip (5mm) is such that the anterior edge of the tip meets the Bregma suture (0mm Bregma level). The second injury was induced at the same location. This impact was previously characterized such that a single injury would result in minimal gliosis or cell death without resulting in skull fracture48. The scalp was sutured using vicryl sutures containing antibiotics (Ethicon, Cincinnati, OH). Sham-injured animals underwent identical anesthesia and surgical procedures without receiving an impact on the final day of CHI. All mice were monitored on a heating pad until they became ambulatory. Additionally, mice were evaluated at 13h and 24h after each injury, followed by daily inspections. All mice were required to maintain 85% of their starting weight in order to receive repeated head injury. However, no mice needed to be removed from the study. After euthanasia at 48h post-injury, mitochondria were extracted by either by DC (based on previous studies31,47), UC (as in 1.7), or FMMS (as in 1.8). For UC methods, brain regions were pooled from three mice for one data point to achieve a measurable synaptic mitochondria fraction. Additionally, respiration assays were completed according to Seahorse XFe24 methods detailed above.

Mitochondrial fractions, obtained using UC or FMMS protocols, were lysed in RIPA buffer (Cat# R0268) and protein concentration determined using the Pierce BCA Protein Assay Kit (ThermoFisher). Equal amounts of proteins were then separated using 416% Mini-PROTEAN TGXTM precast gels (Cat# 4561106, Bio-Rad). After transferring onto nitrocellulose membranes, the blots were incubated with 5% nonfat milk for 12hr before an overnight incubation with primary antibodies at 4C. The primary antibodies used included anti-calnexin (Cat # ab22595; Abcam), anti-beta tubulin (Cat # ab6046; Abcam) and NDUAF9 (Cat # 459100, Invitrogen). Blots were then incubated with Affinipure peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG (Cat # 115035166 and 111-035-144, respectively; Jackson ImmunoResearch laboratories, Inc.) secondary antibodies. Immunoreactive signals were visualized on x-ray film using a SuperSignal West Pico PLUS Chemiluminescent Substrate (Cat # 32209, Pierce). The immunoreactive bands on the x-ray film were scanned using ODYSSEY CLX (LI-COR) and immunoreactive signals quantified using Image Studio Ver 5.2.

Statistical analysis was performed using Graph Pad Prism (GraphPad Software, CA, USA). For all analyses, the significance of differences among groups was set at p<0.05. Triplicate data was averaged for each data point. For each measure, data were measured using interval/ratio scales. The Brown-Forsythe and Bartletts tests were performed to ensure homogeneity of variance. Furthermore, the Shapiro-Wilk test was completed to ensure normality. As these criteria were met for all experimental data, parametric statistics were employed for all data analyses. For all mitochondrial assessments, data sets were evaluated using an unpaired t-test to determine significance between the groups.

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The authors would like to thank Malinda Spry, Binoy Joseph, Ph.D., Jennifer Gooch, and Hemendra Vekaria, Ph.D. for their technical assistance. We would also like to acknowledge the Biomedical Illustration team of Matt Hazzard and Tom Dolan in University of Kentucky Information Technology. This work was supported by NSF EPSCoR Seed Grant 4978/111315 (National Science Foundation Grant No. 1539068), Kentucky Spinal Cord and Head Injury Research Trust (KSCHIRT) Grant 1413A, and VA Merit Award 1I01BX003405-01A1.

All authors had full access to the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Conceptualization, W.B.H., C.L.H., and P.G.S.; Methodology, W.B.H., C.L.H., and P.G.S. Investigation, W.B.H., P.P., and C.L.H.; Formal Analysis, W.B.H. Resources, K.E.S, J.E.S. and P.G.S.; Writing - Original Draft, W.B.H. and C.L.H.; Writing - Review & Editing, W.B.H., K.E.S., J.E.S., and P.G.S.; Visualization, W.B.H.; Supervision, P.G.S.; Funding Acquisition, W.B.H., K.E.S. and P.G.S.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the articles Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the articles Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Hubbard, W.B., Harwood, C.L., Prajapati, P. et al. Fractionated mitochondrial magnetic separation for isolation of synaptic mitochondria from brain tissue. Sci Rep 9, 9656 (2019). https://doi.org/10.1038/s41598-019-45568-3

magnetic separation technology for a recycling industry

magnetic separation technology for a recycling industry

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