Name: preparation of synaptoneurosomes from mouse cortex using a discontinuous percoll-sucrose density gradient
Uploaded: 2011-09-17T14:00:00.000+00:00
Duration: 8 min 30 s
Description: Watch this Scientific Journal Video about Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient at JoVE.com

ברצוננו להודות BK אוגוסט מאוניברסיטת מתקן אלקטרונים וויסקונסין מיקרוסקופ עבור במיקרוסקופ אלקטרונים. עבודה זו נתמכה על ידי NIH מענקים R01-DA026067 ו-P30 HD03352 (עד JSM).

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אין ניגודי אינטרסים הכריז.

הכנה רציפה Percoll-סוכרוז שיפוע המתוארים כאן היא שיטה מהירה, אמינה לבודד SNS פעיל, אשר ניתן להשתמש במגוון של ניסויים בשידור סינפטי. שיטה זו שיפוע, אשר מבוססת על שיטה שפותחה על ידי Dunkley et al. 3,4 הוא המוח subcellular חלוקה הליך המבודדת הן טרום postsynaptic קרום הנגזרות שלפוחית ​​הקש...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> שם מגיב </th><th> חברה </th><th> מספר קטלוגי </th><th> הערות (אופציונלי) </th></tr><tr><td> מיקרו Assay BCA חלבון Kit </td><td> לנקב </td><td> 23235 </td><td></td></tr><tr><td> CaCl 2 </td><td> דיג </td><td> C79-500 </td><td></td></tr><tr><td> CO 2 גז </td><td> Airgas (UW-MDS) </td><td> תקליטור 50 </td><td></td></tr><tr><td> EDTA </td><td> RPI </td><td> E57020 </td><td></td></tr><tr><td> EtOH </td><td> דיג </td><td> A407SK-4 </td><td></td></tr><tr><td> HCl </td><td> דיג </td><td> A142-212 </td><td></td></tr><tr><td> Percoll </td><td> GE Healthcare </td><td> 17-0891-01 </td><td></td></tr><tr><td> KH 2 PO 4 </td><td> דיג </td><td> P285-500 </td><td></td></tr><tr><td> PierceSDS-PAGE Prep ערכת דוגמאות </td><td> לנקב </td><td> 89888 </td><td></td></tr><tr><td> NaCl </td><td> RPI </td><td> S23020 </td><td></td></tr><tr><td> NaHCO 3 </td><td> דיג </td><td> BP328-500 </td><td></td></tr><tr><td> Na Pho 2 4 </td><td> דיג </td><td> S381-500 </td><td></td></tr><tr><td> סוכרוז </td><td> RPI </td><td> S24060 </td><td></td></tr><tr><td> מאגר טריס </td><td> RPI </td><td> T60040 </td><td></td></tr><tr><td> Tetrodotoxin </td><td> סיגמא </td><td> T5651 </td><td></td></tr><tr><td> לייבל Pro Express Mix S35 תג קל </td><td> פרקין אלמר </td><td> NEG772 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><th> ציוד </th><th> חברה </th><th> מספר קטלוגי </th><th> הערות (אופציונלי) </th></tr><tr><td> Dissection כלים </td><td></td><td></td><td></td></tr><tr><td> Dounce homogenizer, 7 מ&quot;ל (מגיע עם זכוכית two pestles שכותרתו &quot;A&quot; ו - &quot;B&quot;) </td><td> ויטון </td><td></td><td></td></tr><tr><td> P1000 Gilson Pipetman </td><td> Gilson </td><td> F123602 </td><td></td></tr><tr><td> אלגרה צנטריפוגה 6KR </td><td> Beckman Coulter </td><td> 366830 </td><td></td></tr><tr><td> GH 3.8 הרוטור, הרוטור דלי Swinging </td><td> Beckman Coulter </td><td> 360581 </td><td></td></tr><tr><td> Beckman J2-21 צנטריפוגה </td><td> Beckman </td><td></td><td></td></tr><tr><td> Beckman צינורות עם כובעים </td><td> Beckman </td><td> 355672 </td><td></td></tr><tr><td> מתאמי קירות לבנים </td><td> Beckman </td><td> 342327 </td><td></td></tr><tr><td> מתאמי חומה כחול </td><td> Beckman </td><td></td><td></td></tr><tr><td> JA-17 רוטור, קבוע זווית הרוטור </td><td> Beckman </td><td> 369691 </td><td></td></tr></tbody></table> טבלה של נוגדנים המשמשים כתמים המערבי: <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> שם נוגדן </th><th> חברה </th><th> מספר קטלוגי </th><th> מארח המינים </th><th> דילול פקטור </th></tr><tr><td> β-אקטין </td><td> סיגמא </td><td> A5441 </td><td> עכבר </td><td> 1:2000 </td></tr><tr><td> GFAP </td><td> סנטה קרוז </td><td> SC-65343 </td><td> עכבר </td><td> 1:200 </td></tr><tr><td> GP73 </td><td> סנטה קרוז </td><td> SC-134509 </td><td> ארנב </td><td> 1:200 </td></tr><tr><td> HSC70 </td><td> סנטה קרוז </td><td> SC-7298 </td><td> עכבר </td><td> 1:200 </td></tr><tr><td> Laminβ </td><td> סנטה קרוז </td><td> SC-6261 </td><td> עז </td><td> 1:200 </td></tr><tr><td> Prohibitin </td><td> סנטה קרוז </td><td> SC-28259 </td><td> ארנב </td><td> 1:200 </td></tr><tr><td> PSD95 </td><td> Millipore </td><td> MAB1596 </td><td> עכבר </td><td> 5 מיקרוגרם / μL </td></tr><tr><td> SNAP25 </td><td> AbCam </td><td> ab5666-100 </td><td> ארנב </td><td> 1:2000 </td></tr><tr><td> Synaptophysin </td><td> Millipore </td><td> MAB368 </td><td> עכבר </td><td> 1:500 </td></tr><tr><td> β-3-טובולין </td><td> סנטה קרוז </td><td> SC-80016 </td><td> עכבר </td><td> 1:200 </td></tr></tbody></table>

preparation of synaptoneurosomes from mouse cortex using a discontinuous percoll-sucrose density gradient

Synaptoneurosomes (SNS) מתקבלים לאחר homogenization ועל חלוקה של קליפת המוח העכבר. הם resealed שלפוחית ​​או מסופי מבודדים להתנתק מסופי האקסון כאשר רקמת קליפת המוח הוא הומוגני. SNS לשמור על המאפיינים טרום postsynaptic, מה שהופך אותם שימושיים בחקר בשידור סינפטי. הם שומרים על מנגנון מולקולרי המשמש איתות עצבי ומסוגלים ספיגת, אחסון, שחרור של נוירוטרנסמיטורים. ההפקה והבידוד של פעיל SNS יכול להיות בעייתי כמו שימוש במדיות Ficoll, אשר ניתן ציטוטוקסיות ודורשים צנטריפוגה המורחבת עקב צפיפות גבוהה, סינון ושיטות צנטריפוגה, אשר עלולה לגרום פעילות נמוכה עקב נזק מכני של SNS. עם זאת, השימוש Percoll-סוכרוז הדרגתיים רציפה צפיפות לבודד SNS מספק שיטה מהירה כדי לייצר תשואות טובות של SNS פעיל translationally. השיטה Percoll-סוכרוז שיפוע הוא מהיר ועדין כפי שהיא מעסיקה התנאים איזוטוני, יש ספינים צנטריפוגה פחות קצר נמנע צעדים צנטריפוגה כי גלולה SNS ולגרום נזק מכני.

Watch this Scientific Journal Video about Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient at JoVE.com

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

שיטה להכין פעיל translationally, synaptoneurosomes שלם (SNS) של קליפת המוח עכבר מתואר. השיטה משתמשת שיפוע רציפה Percoll-סוכרוז צפיפות המאפשרת הכנה מהירה של פעיל SNS.

הכנת Synaptoneurosomes מ - עכבר Cortex באמצעות צבע רציפה Percoll-סוכרוז צפיפות

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

preparation-synaptoneurosomes-from-mouse-cortex-using-discontinuous

Research

JoVE Journal

Neuroscience

31.5K Views.  University of Wisconsin. שיטה להכין פעיל translationally, synaptoneurosomes שלם (SNS) של קליפת המוח עכבר מתואר. השיטה משתמשת שיפוע רציפה Percoll-סוכרוז צפיפות המאפשרת הכנה מהירה של פעיל SNS.

Video: Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

קרום המוח Fractation: An<em&gt; אקס ויוו</em&gt; גישה כדי להעריך לוקליזציה חלבון subsynaptic

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

Biochemistry

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

הכנת ניתק תרבויות עכבר קליפתיים Neuron

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Biology

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960&#8217;s, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

הכנת Synaptic פלזמה ממברנה וחלבוני צפיפות Postsynaptic באמצעות שיפוע סוכרוז רציף

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

Peering at Brain Polysomes with Atomic Force Microscopy

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

מציץ המוח Polysomes עם מיקרוסקופית כוח אטומי

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

גבוהה פרופיל Proteome Synaptic החלטה כמותי של אזורים במוח העכבר לאחר למידה אפליה שמיעתית

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

אפיון ובידוד של העכבר מיקרוגלייה העיקרי על ידי צנטריפוגה הדרגתיות צפיפות

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell types. This balance is tightly controlled by gene expression that is fine-tuned at multiple levels, including transcription, post-transcription and translation. In this regard, a growing body of evidence highlights a critical role of translational regulation in coordinating neural stem cell fate decisions. Polysome fractionation is a powerful tool for the assessment of mRNA translational status at both global and individual gene levels. Here, we present an in-house polysome profiling pipeline to assess translational efficiency in cells from the developing mouse cerebral cortex. We describe the protocols for sucrose gradient preparation, tissue lysis, ultracentrifugation and fractionation-based analysis of mRNA translational status.

The development of the mammalian brain requires proper control of gene expression at the level of translation. Here, we describe a polysome profiling system with an easy-to-assemble sucrose gradient-making and fractionation platform to assess the translational status of mRNAs in the developing brain.

ניתוח תרגום במוח העכבר המתפתח באמצעות פרופיל פוליזום

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell ...

Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological disorders. The characterization of synaptic sub-compartments by biochemical isolation is, therefore, a powerful method to elucidate the molecular bases of synaptic processes, both in health and disease. This protocol describes the isolation of synaptic terminals and synaptic sub-compartments from mouse brains by subcellular fractionation. First, sealed synaptic terminal structures, known as synaptosomes, are isolated following brain tissue homogenization. Synaptosomes are neuronal pre- and post-synaptic compartments with pinched-off and sealed membranes. These structures retain a metabolically active state and are valuable for studying synaptic structure and function. The synaptosomes are then subjected to hypotonic lysis and ultracentrifugation to obtain synaptic sub-compartments enriched for synaptic vesicles, synaptic cytosol, and synaptic plasma membrane. Fraction purity is confirmed by electron microscopy and biochemical enrichment analysis for proteins specific to sub-synaptic compartments. The presented method is a straightforward and valuable tool for studying the structural and functional characteristics of the synapse and the molecular etiology of various brain disorders.

This protocol presents a robust, detailed method to obtain highly pure synaptosomes, synaptic vesicles, and other synaptic fractions from the mouse brain. This method enables the evaluation of synaptic processes, including the biochemical analysis of protein localization and function with compartmental resolution.

פיצול תת-תאי לבידוד רכיבים סינפטיים ממוח מורין

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological ...

Mononuclear Cell Isolation from Mouse Brain: A Density Gradient Centrifugation Technique to Isolate Brain Resident Mononuclear Cells

Source: Ji, Z. et al. <a href="https://www.jove.com/v/61050/flow-cytometric-analysis-lymphocyte-infiltration-central-nervous">Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis.</a> J. Vis. Exp. (2020)
In this video, we perform the isolation of mononuclear cells from a mouse&#8217;s brain using density gradient centrifugation. The isolated mononuclear cell suspension can be used for further downstream analysis.

בידוד תאים חד-גרעיניים ממוח עכבר: טכניקת צנטריפוגה של שיפוע צפיפות לבידוד תאים חד-גרעיניים שוכני מוח

Source: Ji, Z. et al. Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis. J. ...

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Cancer Research

Cancers of the Nervous System

Ex vivo studies

Isolation of Microglia from the Cortex of an Adult Mouse Brain

First, use a razor blade to chop each brain region into fine pieces that are less than 1 cubic millimeter. Using a 1-milliliter pipette with the tip cut off, transfer the tissue pieces into pre-chilled Dounce homogenizers. Homogenize the tissue by slowly twisting the piston in and out of the Dounce homogenizer for 6 to 10 full strokes until no visible chunks are present. Then, transfer the dissociated tissues into 50-milliliter tubes through 70-micrometer strainers.
Rinse each Dounce homogenizer and piston with a total of 6 milliliters of cold medium A, then transfer the rinsing to a 15-milliliter tube for centrifugation. Centrifuge single-cell suspension at 400 times g and at 4 degrees Celsius for 5 minutes with a break at 5.
Rinse one large depletion column and three large selection columns in a magnetic separator with 3 milliliters of MCS. When the centrifugation for the tissue samples is complete, pipette out and discard the supernatant without disturbing the pellet. Resuspend the cells in MCS buffer that contains RNase inhibitor.
Add 100 microliters of myelin removal beads to each tube containing resuspended cells from the cortex and cerebellum, and add 50 microliters of myelin removal beads to each of the tubes containing resuspended cells from the hippocampus and striatum. Incubate the tubes on ice for 10 minutes.
After this, add MCS to the tube containing cortical cells to bring its volume up to 2 milliliters, and to the remaining tubes to bring each of their volumes up to 1 milliliter. Once the columns are empty of rinsing buffer, load 2 milliliters of cortical cells onto the large depletion column and 1 milliliter of each other cell suspension onto separate large selection columns. Next, wash the large depletion column once with 1 milliliter of MCS buffer, and wash each large selection column twice using 1 milliliter of MCS buffer for each wash.

הכנת Synaptoneurosomes מ - עכבר Cortex באמצעות צבע רציפה Percoll-סוכרוז צפיפות

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קרום המוח Fractation: An