Wir erkennen den Beitrag von Renata Sano in der Konzeption des ersten Protokolls. Ad&#39;A. hält die Jewelers Für Kinder (JFC) Stiftungsprofessur für Genetik und Gentherapie. Diese Arbeit wurde zum Teil durch NIH Zuschüsse GM60905, DK52025 und CA021764, und den amerikanischen libanesischen syrischen Assoziierte Charities (ALSAC) finanziert.

<html> Das folgende Protokoll ist für die Isolierung und Aufreinigung von MAMs und Edelsteine ​​aus Maushirn bestimmt Lösungen für die Isolierung von Mitochondrien, MAMs und Edelsteine ​​benötigt Fraktionierung, um rohes mitochondrialen Präparation zu erhalten: Lösung A: 0,32 M Saccharose, 1 mM NaHCO 3, 1 mM MgCl 2, 0,5 mM CaCl 2 + Protease-Inhibitoren (add frisch, je nach Bedarf) Lösung B: 0,32 M Saccharose, 1 mM NaHCO 3 + Protease-Inhibitoren (add frisch, je nach Bedarf) MAMs Isolation: Isolierung Medium: 250 mM Mannit, 5 mM HEPES, pH 7,4, 0,5 mM EGTA, 0,1% BSA Gradient Puffer: 225 mM Mannitol, 25 mM HEPES pH 7.5, 1 mM EGTA, 0,1% BSA (Endkonzentration) GEMs Isolation: Inhalt &quot;&gt; GEM Extraction Buffer: 25 mM HEPES pH 7,5, 0,15 M NaCl, 1% Triton X-100 + Protease-Inhibitoren (add frisch, je nach Bedarf) GEM solubilisierende Puffer: 50 mM Tris-HCl, pH 8,8, 5 mM EDTA, 1% SDS + Protease-Inhibitoren (add frisch, je nach Bedarf). Ein. First Step subzelluläre Fraktionierung: Entfernen der Kerne, lysierten Zellen und Zelltrümmer <ol><li> Einschläfern Maus in CO 2-Kammer. </li><li> Umgehend entfernen Gehirn, halbieren, legen in 2 ml Röhrchen auf Eis und wiegen. </li></ol> Hinweis: Bewahren Sie Gehirnhälften separaten während des gesamten Verfahrens. Das gilt für die Behandlung von einem einzigen Gehirn Hälfte. <ol start="3"><li> Die Hälfte Gehirn in einer vorgekühlten 2 ml Glas Dounce Gewebe Mahlwerk mit 1 ml kaltem Lösung A. mit insgesamt 15 Schlägen eines großen Freiraum Pistill homogenisiert. </li><li> Transfer zu einem 15 ml Falcon-Röhrchen auf Eis und verdünnterHomogenate bis zu 10 Volumina w / v, z. B. 0,225 g bis 2,25 ml. </li><li> Zentrifuge Proben bei 1.400 xg für 10 min bei 4 ° C. </li><li> Entfernen Sie vorsichtig den Überstand und Transfer zu einem 30 ml-Rundkolben aus Glas Zentrifugenröhrchen auf Eis. Achten Sie darauf, um das Pellet zu stören. </li><li> Resuspendieren des Pellets in der gleichen 10 Volumen Lösung A. Homogenisieren in derselben Schleifmaschine, mit 3-6 Striche eines kleinen Zwischenraum Pistill, 1 ml auf einmal. </li><li> Übertragen zu einem frischen 15 ml Falcon-Röhrchen und Zentrifuge bei 710 × g für 10 min bei 4 ° C. Dies führt zu einem Pellet von Kerne und Zelltrümmer. </li><li> Entfernen Sie vorsichtig den Überstand und Pool mit dem Überstand in Schritt 1,6 gespeichert. </li></ol> 2. Zweiter Schritt: Crude Mitochondrien Isolation <ol><li> Zentrifuge Überstände bei 13.800 × g für 10 min bei 4 ° C. </li><li> Überstand in ein Ultra-Clear Beckman Zentrifugenröhrchen auf Eis </li><li> Pellet in 10 Volumen Lösung A. Homogenisieren in derselben Schleifmaschine, mit 3-6 Striche eines kleinen Zwischenraum Pistill, 1 ml auf einmal. </li><li> Zentrifuge nach Schritt 2.1. </li><li> Wiederholen Sie die Schritte 2,2-2,4. </li><li> Das resultierende Pellet ist eine angereicherte mitochondrialen Fraktion. Pool die Überstände (Cytosol und ER), mit Parafilm abdecken und auf Eis. </li><li> Das Pellet mit 6 Striche eines kleinen Zwischenraum Pistill in 4,8 ml / g (g wird auf die ursprüngliche Gehirngewicht bezeichnet) der Lösung B, in einer frischen Homogenisator. </li><li> Verwendung von Glas Pasteurpipetten, bereiten einen diskontinuierlichen Sucrosegradienten in einem Ultra-Clear Beckman Zentrifuge Rohr, das Hinzufügen anschließend dem Boden des Röhrchens wie folgt, sorgfältig sichergestellt keine Blasen gebildet werden: Resupended Pellet (0,32 M Saccharose) 3 ml 850 mM Saccharose (in 1 mM NaHCO 3) 3 ml 1 M Saccharose (in 1 mM NaHCO 3) 3 ml 1,2 M Sucrose (in 1 mM NaHCO3) </li><li> Zentrifugieren bei 82.500 × g für 2 Stunden bei 4 ° C. Die resultierende Trennung am Ende des Gradienten wird erzeugen drei Bands und ein Pellet: 1) Myelin und anderen Kontaminanten Membran (0,32 M - 0,85 M-Schnittstelle), 2) ER, Golgi, Plasmamembranen (0,85 M - 1 M-Schnittstelle); 3) Synaptosomen (1 M - 1,2 M-Schnittstelle), 4) Rohprodukt Mitochondrien (Pellet), das für die nachfolgenden Reinigungsstufen verwendet </li></ol> 3. Isolierung von Mitochondrien-assoziierte ER Membran, MAMs <ol><li> Resuspendieren des frisch isolierten rohen Mitochondrien von einer Gehirnhälfte, in 2 ml Isolation Medium mit frisch hinzugefügten Protease-Inhibitoren. </li><li> Bereiten Sie eine 30% Percoll Gradienten mit Gradient Puffer 8 ml pro halbe Gehirn, und in einem Ultra-Clear Beckman Zentrifuge Tube. </li></ol> * Hinweis: Achten Gradientenpuffer konzentrierter (1,43 mal), um die korrekte Endkonzentration nach d zu erreicheniluting Percoll und 30%. <ol start="3"><li> Layer mitochondriale Suspension auf der vorbereiteten Gradienten langsam zu vermeiden Blasen. </li><li> Zentrifugieren bei 95.000 × g für 30 min, 4 ° C. </li><li><ol class="lroman"><li> Entfernen Sie die schwere Fraktion (unteres Band) FIRST mit einem Glas Pasteur Pipette und Transfer zu einem frischen Rundkolben Glasrohr, auf Eis. </li><li> Entfernen Sie die leichte Fraktion (oberes Band) SECOND mit einem anderen Glas Pasteur Pipette und Transfer zu einem separaten frischen Rundkolben Glasrohr, auf Eis. </li></ol></li><li> Verdünnte beide Fraktionen in Schritt 3.5 mit 10 ml Isolationsmedium und Zentrifuge gesammelt bei 6.300 xg für 10 min bei 4 ° C. </li><li> Verwerfen des Überstands aus der schweren Fraktion in Schritt 3,6 erhalten wird, das Pellet mit weiteren 10 ml Isolationsmedium und zentrifugieren, wie in Schritt 3.6. Das resultierende Pellet wird die reine mitochondriale Fraktion sein. </li><li> Den Überstand aus dem Licht Fraction in Schritt 3,6 bis einem Ultra-Clear Beckman Zentrifugenröhrchen erhalten, auf Eis. Entsorgen Sie die Pellets. </li><li> Hinzufügen ausreichender Isolation Medium zu dem Überstand in Schritt erhaltenen 3,8, um das Rohr und Zentrifuge bei 100.000 × g für 1 Stunde, 4 ° C zu füllen Das resultierende Pellet wird die MAMs Bruch sein. Entfernen Sie vorsichtig und den Überstand verwerfen. </li></ol> Hinweis: Bei diesem Schritt können Sie auch bei 100.000 xg zentrifugieren für 1 Stunde, 4 ° C wird der Überstand in Schritt 2,6 gespeichert. Das Pellet wird der ER-Fraktion sein, und der Überstand der cytosolischen Fraktion. 4. Extraktion von Glycosphingolipid angereichert Mikrodomänen, GEMs <ol><li> Lyse pure Mitochondrien und / oder MAM Fraktionen in 500 ul-1 ml Extraktionspuffer für 20 min auf Eis. </li><li> Zentrifuge Lysaten bei 15.300 × g für 2 Minuten bei 4 ° C. </li><li> Sammle Überstände (Triton X-100 löslichen Material) und re-Zentrifuge Pelletsfür 2 min zu entfernen verbleibende lösliche Material. (Triton X-100 extrahiert Mitochondrien und / oder Triton X-100 extrahiert MAMs). </li><li> Solubilisierung Pellets in Lösungsvermittler Buffer. Diese solubilisierten Materials stellt die GEM Fraktionen und können zur weiteren Analyse verwendet werden. </li></ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+synthesis+in+a+membrane+fraction+associated+with+mitochondria.">Phospholipid synthesis in a membrane fraction associated with mitochondria.</a> J. Biol. Chem. 265, 7248-7256 (1990).</li><li>Wieckowski, M. R., Giorgi, C., Lebiedzinska, M., Duszynski, J., Pinton, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mitochondria-associated+membranes+and+mitochondria+from+animal+tissues+and+cells.">Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells.</a> Nat. 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Keiner der Autoren keine Interessenkonflikte zu erklären.

Die Stellen des Kontakts zwischen intrazellulären Membranen oder zwischen Organellen und der Plasmamembran von Zellen repräsentieren dynamischen Signalisierung Plattformen für grundlegende zelluläre Prozesse. Die genaue Charakterisierung ihrer Funktion und Zusammensetzung sowohl unter physiologischen und pathologischen Bedingungen erfordert zuverlässige und reproduzierbare Reinigung Protokolle. Die Verfahren detailliert hier wurden speziell von unserem Labor zur Isolierung und Aufreinigung der MAMs und ihre entspre...

<table border="1"><tbody><tr><td> Name des Reagenz / Material </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td></td><td></td><td></td><td> REAGENZIEN </td></tr><tr><td></td><td></td><td></td><td> Fraktionierung </td></tr><tr><td> Saccharose </td><td> Fisher Scientific </td><td> S5-500 </td><td></td></tr><tr><td> Sodium Bicarbonate </td><td> Sigma-Aldrich </td><td> S-5761 </td><td></td></tr><tr><td> Magnesiumchlorid, Hexahydrate </td><td> Fisher Scientific </td><td> BP214-500 </td><td></td></tr><tr><td> Calciumchlorid, Dihydrat </td><td> Sigma-Aldrich </td><td> C-5080 </td><td></td></tr><tr><td></td><td></td><td></td><td> MAM </td></tr><tr><td> D-Mannitol </td><td> Sigma-Aldrich </td><td> M9546-250G </td><td></td></tr><tr><td> Hepes </td><td> Fisher Scientific </td><td> BP310-500 </td><td></td></tr><tr><td> EGTA </td><td> Sigma-Aldrich </td><td> E4378-250G </td><td></td></tr><tr><td> BSA, Fraktion V, Heat Shock, Lyophilisat </td><td> Roche </td><td> 03-116-964-001 </td><td></td></tr><tr><td> Percoll </td><td> GE </td><td> 17-0891-02 </td><td></td></tr><tr><td></td><td></td><td></td><td> GEM </td></tr><tr><td> Triton X-100 </td><td> Sigma-Aldrich </td><td> T9284-500 ml </td><td></td></tr><tr><td> Kochsalz </td><td> Fisher Scientific </td><td> S271-3 </td><td></td></tr><tr><td> Tris Base </td><td> Roche </td><td> 03-118-142-001 </td><td></td></tr><tr><td> HCl </td><td> Fisher Scientific </td><td> A144S-500 </td><td></td></tr&gt; <tr><td> EDTA </td><td> Fisher Scientific </td><td> BP120-500 </td><td></td></tr><tr><td> Natriumdodecylsulfat (SDS) </td><td> Fisher Scientific </td><td> BP166-500 </td><td></td></tr><tr><td></td><td></td><td></td><td> Gemeinsam </td></tr><tr><td> Protease-Inhibitoren Tablets, Complete EDTA-free </td><td> Roche </td><td> 11-873-580-001 </td><td></td></tr><tr><td></td><td></td><td></td><td> EQUIPMENT </td></tr><tr><td> 2 ml Douce All-Glass Tissue Grinders </td><td> Kimble Chase </td><td> 885300-0002 </td><td></td></tr><tr><td> 15 ml Polypropylen Konische Zentrifugenröhrchen, BD Falcon </td><td> BD </td><td> 352097 </td><td></td></tr><tr><td> 30 ml Round-Bottom Glass Zentrifugenröhrchen </td><td> Kimble Chase </td><td> 45500-30 </td><td></td></tr><tr><td> 15 ml Round-Bottom Glass Centrifuge Tubes </td><td> Kimble Chase </td><td> 45500-15 </td><td></td></tr><tr><td> Ultrazentrifuge Röhren, Ultra-Clear, Thinwall, 14x89 mm </td><td> Beckman-Coulter </td><td> 344059 </td><td></td></tr><tr><td> Parafilm </td><td> Cole-Parmer </td><td> PM996 </td><td></td></tr><tr><td> Einweg-Flaschen aus Glas Pasteur Pipetten, 9 &quot; </td><td> Fisher Scientific </td><td> 13-678-20C </td><td></td></tr></tbody></table>

mitochondria-associated er membranes (mams) and glycosphingolipid enriched microdomains (gems): isolation from mouse brain

Intrazellulären Organellen sind hochdynamische Strukturen mit unterschiedlicher Form und Zusammensetzung, die Zell-spezifischen intrinsischen und extrinsischen Hinweise ausgesetzt sind. Ihre Membranen werden häufig an definierten Kontaktstellen, die Naben für den Austausch von Signalmolekülen und Membrankomponenten 1,2,3,4 geworden gegenübergestellt. Die miteinander organellären Membranmikrodomänen, die zwischen dem endoplasmatischen Retikulum (ER) und den Mitochondrien bei der Öffnung des IP3-Ca 2 +-Kanal als den Mitochondrien-ER zugehörigen Membranen oder MAMs 4,5,6 bekannt sind ausgebildet sind. Das Protein / Lipid-Zusammensetzung und biochemischen Eigenschaften dieser Membran Kontaktstellen wurden ausgiebig insbesondere in Bezug auf ihre Rolle bei der Regulierung der intrazellulären Ca 2 + 4,5,6 untersucht. Die ER dient als primären Speicher von intrazellulärem Ca 2 +, und reguliert in diesem Rahmen eine Vielzahl von zellulären Prozessen stromabwärts von Ca 2 +-Signalisierung, inkl.uding posttranslationale Proteinfaltung und Protein maturation7. Mitochondrien, auf der anderen Seite aufrechtzuerhalten Ca 2 +-Homöostase durch Puffern zytosolischen Ca 2 +-Konzentration wodurch die Einleitung Apoptosewege stromabwärts von Ca 2 + 4,8 Unwucht. Der dynamische Charakter der MAMs macht sie zu idealen Standorte, um grundlegende zelluläre Mechanismen, einschließlich Ca 2 +-Signalisierung und Regulation der mitochondrialen Ca 2 +-Konzentration, Lipid-Biosynthese und Transport, Energiestoffwechsel und das Überleben der Zelle 4,9,10,11,12 sezieren. Mehrere Protokolle wurden für die Reinigung dieser Mikrodomänen aus Lebergewebe und kultivierten Zellen 13,14 beschrieben. Unter zuvor publizierten Methoden in Betracht, haben wir ein Protokoll für die Isolierung von Mitochondrien und MAMs vom erwachsenen Mäusehirn angepasst. Um dieses Verfahren haben wir eine zusätzliche Reinigungsstufe, nämlich ein Triton X100 Extraktion hinzugefügt, die enables die Isolierung des Glycosphingolipid angereicherten Mikrodomäne (GEM) Bruchteil der MAMs. Diese Zubereitungen GEM teilen sich mehrere Proteinkomponenten mit Caveolae und Lipidflößen, aus der Plasmamembran oder anderen intrazellulären Membranen abgeleitet und vorgeschlagen, als Sammelpunkte für die Bündelung von Rezeptorproteinen und für Protein-Protein-Wechselwirkungen 4,15 funktionieren.

Basierend auf unserer Erfahrung mit der Verwendung dieses Protokoll können wir sicher empfehlen es für die Isolierung und Reinigung von MAMs, Edelsteine ​​und mitochondriale Fraktionen aus Mäusehirn. Das Verfahren wie oben in hohem Maße reproduzierbar und konsistent. In Abbildung 1 zeigen wir ein repräsentatives Bild des Weges pure Mitochondrien und MAMs Schicht auf einem Percoll-Gradienten (Schritt 3,4). Eine definierte, milchige Band enthaltenden gereinigten Mitochondrien (Mito-P) segregiert ...

Watch this Scientific Journal Video about Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain at JoVE.com

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

Dieser Vorgang zeigt, wie aus dem erwachsenen Gehirn der Maus die Mitochondrien-assoziierte ER Membranen oder MAMs und den Glykosphingolipid angereicherten Mikrodomänen Fraktionen aus MAMs und mitochondrialen Vorbereitungen zu isolieren.

Mitochondrien-assoziierte ER Membranen (MAMs) und Glycosphingolipid Angereichert Mikrodomänen (GEM): Isolation aus Mäusehirn

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

mitochondria-associated-er-membranes-mams-glycosphingolipid-enriched

Research

JoVE Journal

Neuroscience

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

24.2K Aufrufe.  St Jude Children's Research Hospital. Dieser Vorgang zeigt, wie aus dem erwachsenen Gehirn der Maus die Mitochondrien-assoziierte ER Membranen oder MAMs und den Glykosphingolipid angereicherten Mikrodomänen Fraktionen aus MAMs und mitochondrialen Vorbereitungen zu isolieren.

Serie: Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Isolierung und Kultivierung von Endothelzellen aus dem embryonalen Vorderhirn

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Forschung

Neurowissenschaften

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Isolierung der zerebralen Kapillaren aus frischem menschlichen Hirngewebe

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

Isolation und RNA-Extraktion von Neuronen, Makrophagen und Mikroglia aus Larven Zebrafisch Gehirne

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

Isolierung von regionsspezifischer Mikroglia von einer Erwachsenenmaus Gehirnhälfte für tiefe einzellige RNA-Sequenzierung

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Studying Pre-formed Fibril Induced &amp;#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

Studium der vorgeformten Fibril-induzierten Synuclein-Akkumulation in primären embryonalen Maus-Midbrain-Neuronen

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

Dissektion und Isolierung von murinen Glia aus mehreren Regionen des zentralen Nervensystems

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

Ein mit Fibrin angereichertes und tPA-sensitives photothrombotisches Schlaganfallmodell

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent ...

Quantifizierung der Histonmodifikation des Mausgehirns mittels Durchflusszytometrie

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to histone tails. Histone post-translational modifications (HPTMs) can either facilitate gene expression or repression. For example, acetylation of histone tail lysine residues neutralizes the positive charge and reduces interactions between the tail and negatively charged DNA. The decrease in histone tail-DNA interactions results in increased accessibility of the underlying DNA, allowing for increased transcription factor access. The acetylation mark also serves as a recognition site for bromodomain-containing transcriptional activators, together resulting in enhanced gene expression. Histone marks can be dynamically regulated during cell differentiation and in response to different cellular environments and stimuli. While next-generation sequencing approaches have begun to characterize genomic locations for individual histone modifications, only one modification can be examined concurrently. Given that there are hundreds of different HPTMs, we have developed a high throughput, quantitative measure of global HPTMs that can be used to screen histone modifications prior to conducting more extensive genome sequencing approaches. This protocol describes a flow cytometry-based method to detect global HPTMs and can be conducted using cells in culture or isolated cells from in vivo tissues. We present example data from isolated mouse brain microglia to demonstrate the sensitivity of the assay to detect global shifts in HPTMs in response to a bacteria-derived immune stimulus (lipopolysaccharide). This protocol allows for the rapid and quantitative assessment of HPTMs and can be applied to any transcriptional or epigenetic regulator that can be detected by an antibody.

Autor im Rampenlicht: Verbesserungen in der Forschung zur Genexpressionsregulation

This work describes a protocol for the quantification of global histone modifications using intranuclear flow cytometry in isolated brain microglia. The work also contains the microglia isolation protocol that was used for data collection.

Quantifizierung globaler posttranslationaler Histonmodifikationen mittels intranukleärer Durchflusszytometrie in isolierten Mikroglia des Mausgehirns

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to ...