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).

<ol>
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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

31.2K 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

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

הדמיה כרונית של עכבר-Visual Cortex שימוש ההכנה סחוט הגולגולת

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

הצמד תיקון הקלטות מתא עצב עכבר רשתית בהכנת Dark המותאמות פרוסה

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

בידוד LacZ-להביע תאים פנימית עכבר רקמות האוזן באמצעות זרימת cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

להדמיה מניפולציה גנטית של דנדריטים עם קוצים בקליפת עכבר היפוקמפוס מוחין באמצעות<em&gt; ברחם</em&gt; Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

הדמיה חיה של מיטוזה בCortex עכבר עוברי פיתוח

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

שני פוטונים<em&gt; In vivo</em&gt; הדמיה של קוצים הדנדריטים בCortex העכבר באמצעות דלילה, גולגולת הכנה

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

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 ...

Investigating the Function of Deep Cortical and Subcortical Structures Using Stereotactic Electroencephalography: Lessons from the Anterior Cingulate Cortex

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure involves the chronic placement of multiple depth electrodes into regions of the brain typically inaccessible via subdural grid electrode placement. SEEG thus provides a unique opportunity to investigate brain function. In this paper we demonstrate how SEEG can be used to investigate the role of the dorsal anterior cingulate cortex (dACC) in cognitive control. We include a description of the SEEG procedure, demonstrating the surgical placement of the electrodes. We describe the components and process required to record local field potential (LFP) data from consenting subjects while they are engaged in a behavioral task. In the example provided, subjects play a cognitive interference task, and we demonstrate how signals are recorded and analyzed from electrodes in the dorsal anterior cingulate cortex, an area intimately involved in decision-making. We conclude with further suggestions of ways in which this method can be used for investigating human cognitive processes.

Stereotactic Electroencephalography (SEEG) is an operative technique used in epilepsy surgery to help localize seizure foci. It also affords a unique opportunity to investigate brain function. Here we describe how SEEG can be used to investigate cognitive processes in human subjects.

חוקרים את הפונקציה של עומק קליפת מוח ומבנים קורטיקליים שימוש stereotactic Electroencephalography: לקחים מCortex Anterior cingulate

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

חיים הדמיה של קליפת המוח ראשי תאים באמצעות מערכת תרבות 2D

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

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 ...