Riconosciamo il contributo di Renata Sano nel concepire il protocollo iniziale. Ad&#39;A. detiene le Jewelers per i bambini (JFC) Dotato Cattedra di Genetica e terapia genica. Questo lavoro è stato finanziato in parte da sovvenzioni NIH GM60905, DK52025 e CA021764, e gli americani libanesi Charities siriane associati (ALSAC).

<html> Il seguente protocollo è stato progettato per l&#39;isolamento e la purificazione di MAMs e gemme da cervello di topo Soluzioni richiesto per l&#39;isolamento di mitocondri, MAMs e gemme Frazionamento per ottenere greggio preparazione mitocondriale: Soluzione A: 0,32 M saccarosio, 1 mM NaHCO 3, 1 mM MgCl 2, 0.5 mM CaCl 2 + inibitori delle proteasi (aggiungere fresco, se necessario) Soluzione B: 0.32 M saccarosio, 1 mM NaHCO3 + inibitori della proteasi (aggiungere fresco, se necessario) MAMs Isolamento: Isolation Medio: 250 mM Mannitolo, 5 mM HEPES pH 7,4, 0,5 mM EGTA, 0,1% BSA Gradiente Buffer: 225 mM mannitolo, 25 mM HEPES pH 7,5, 1 mM EGTA, 0,1% BSA (concentrazione finale) GEM di isolamento: contenuto &quot;&gt; GEM tampone di estrazione: 25 mM HEPES pH 7,5, 0,15 M NaCl, 1% Triton X-100 + inibitori delle proteasi (aggiungere fresco, se necessario) GEM Buffer solubilizzante: 50 mM Tris-HCl, pH 8,8, 5 mM EDTA, 1% SDS + Inibitori della proteasi (aggiungere fresco, se necessario). 1. Passo Frazionamento subcellulare Primo: Celle rimozione dei Nuclei, non lisate e detriti cellulari <ol><li> Eutanasia mouse nella camera di CO 2. </li><li> Rimuovere immediatamente il cervello, dimezzare, mettere in 2 ml provette in ghiaccio e pesare. </li></ol> Nota: Tenere le metà del cervello separato durante tutta la procedura. Quanto segue si applica al trattamento di un mezzo unico cervello. <ol start="3"><li> Luogo cervello metà in pre-refrigerati 2 smerigliatrice di vetro tessuto Dounce ml contenente 1 ml di soluzione fredda A. Omogeneizzare con 15 colpi totali di un pestello spazio di grandi dimensioni. </li><li> Trasferire in una provetta da 15 ml falcon, su ghiaccio, e diluireomogenati fino a 10 volumi w / v, ad esempio 0,225 g di 2,25 ml. </li><li> Centrifugare campione a 1400 xg per 10 min a 4 ° C. </li><li> Rimuovere con attenzione il supernatante e trasferirlo in una 30 ml a fondo rotondo tubo di vetro, sul ghiaccio. Fare attenzione a non disturbare il pellet. </li><li> Risospendere il precipitato in stessi 10 volumi di soluzione A. Omogeneizzare nel macinacaffè stesso, con 3-6 colpi di un pestello piccolo gioco, 1 ml per volta. </li><li> Trasferire in una nuova provetta 15 ml falcon e centrifugare a 710 xg per 10 min a 4 ° C. Ciò si traduce in un pellet di nuclei e detriti cellulari. </li><li> Rimuovere con attenzione il surnatante e piscina con il surnatante salvato nel passaggio 1.6. </li></ol> 2. Secondo passo: Crude Isolamento mitocondri <ol><li> Centrifugare supernatanti a 13800 xg per 10 min a 4 ° C. </li><li> Trasferire il surnatante in un Ultra-Clear provetta da centrifuga Beckman, su ghiaccio </li><li> Risospendere il pellet in 10 volumi di soluzione A. Omogeneizzare nel macinacaffè stesso, con 3-6 colpi di un pestello piccolo gioco, 1 ml per volta. </li><li> Centrifugare come punto 2.1. </li><li> Ripetere i passaggi 2,2-2,4. </li><li> Il pellet risultante è una frazione arricchita mitocondriale. I supernatanti (citosol e ER), coprire con parafilm e tenere in ghiaccio. </li><li> Risospendere il pellet con 6 colpi di un pestello piccolo gioco, in 4,8 ml / g (g è riferito al peso del cervello originale) di soluzione B, in un omogeneizzatore fresco. </li><li> Usando pipette Pasteur in vetro, preparare un gradiente discontinuo di saccarosio in un Ultra-Clear provetta da centrifuga Beckman, aggiungendo successivamente al fondo della provetta come segue, con attenzione, assicurando bolle si formano: Resupended pellet (0,32 M saccarosio) 3 ml 850 mM saccarosio (in 1 mM NaHCO3) 3 ml 1 M Saccarosio (in 1 mM NaHCO 3) 3 ml di 1,2 M saccarosio (in 1 mM NaHCO3) </li><li> Centrifugare a 82500 xg per 2 ore a 4 ° C. La separazione risultante alla fine del gradiente produrrà tre bande e un pellet: 1) mielina e altri contaminanti membrana (0,32 M - 0,85 M di interfaccia), 2) ER, Golgi, membrane plasmatiche (0,85 M - 1 M di interfaccia); 3) sinaptosomi (1 M - 1,2 M di interfaccia), 4) grezzo mitocondri (pellet) che viene utilizzato per le successive fasi di purificazione </li></ol> 3. Isolamento dei mitocondri associata a membrana ER, MAMs <ol><li> Risospendere il greggio appena mitocondri isolati da una metà del cervello, in 2 ml di terreno di isolamento contenenti inibitori della proteasi appena aggiunti. </li><li> Preparazione di un gradiente Percoll 30% con il tampone di gradiente da 8 ml per metà del cervello, e il luogo in un Ultra-Clear provetta da centrifuga Beckman. </li></ol> * Nota: Assicurarsi tampone gradiente più concentrato (1,43 volte) per ottenere corretta concentrazione finale dopo diluting Percoll al 30%. <ol start="3"><li> Sospensione livello mitocondriale in cima gradiente preparato lentamente per evitare eventuali bolle. </li><li> Centrifugare a 95.000 xg per 30 min a 4 ° C. </li><li><ol class="lroman"><li> Rimuovere la frazione pesante (fascia inferiore) prima con una pipetta Pasteur di vetro e versare in un nuovo fondo rotondo tubo di vetro, su ghiaccio. </li><li> Rimuovere la frazione leggera (banda superiore) con un altro SECONDO Pasteur pipetta di vetro e versare in un separato fresco a fondo rotondo tubo di vetro, su ghiaccio. </li></ol></li><li> Diluire entrambe frazioni raccolte nel passaggio 3,5 con 10 ml di mezzo di isolamento e centrifugare a 6300 xg per 10 min a 4 ° C. </li><li> Scartare il sopranatante della frazione pesante ottenuta nella fase 3.6, risospendere il pellet con un altro supporto 10 ml Isolamento e centrifugare nuovamente, come passo 3,6. Il pellet risultante sarà la frazione pura mitocondriale. </li><li> Trasferire il surnatante dal Frac Lucezione ottenuta nello stadio da 3,6 a un Ultra-Clear provetta da centrifuga Beckman, sul ghiaccio. Eliminare il pellet. </li><li> Aggiungere mezzo di isolamento al supernatante ottenuto nel passaggio 3,8 per riempire il tubo e centrifugare a 100.000 xg per 1 ora, 4 ° C. Il pellet risultante sarà la frazione MAMs. Rimuovere con attenzione ed eliminare il surnatante. </li></ol> Nota: A questo punto, si può anche centrifugare a 100.000 xg per 1 ora, 4 ° C, il surnatante salvato nella fase 2.6. Il pellet sarà la frazione ER e supernatante la frazione citosolica. 4. Estrazione di glicosfingolipidi-microdomini arricchiti, GEM <ol><li> Lyse puri frazioni mitocondri e / o MAM in 500 pl-1 ml di tampone di estrazione per 20 min in ghiaccio. </li><li> Centrifugare a 15300 lisati xg per 2 minuti a 4 ° C. </li><li> Raccogliere surnatanti (Triton X-100 materiale solubile) e ri-centrifuga pelletsper 2 minuti per rimuovere restante materiale solubile. (Triton X-100 mitocondri estratti e / o Triton X-100 MAMs estratti). </li><li> Solubilizzare pellet in solubilizzanti tampone. Questo materiale solubilizzato rappresenta le frazioni GEM e può essere utilizzato per ulteriori analisi. </li></ol>

<ol>
	<li>Poburko, D., Kuo, K. H., Dai, J., Lee, C. H., van Breemen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organellar+junctions+promote+targeted+Ca2++signaling+in+smooth+muscle:+why+two+membranes+are+better+than+one.">Organellar junctions promote targeted Ca2+ signaling in smooth muscle: why two membranes are better than one.</a> Trends Pharmacol. Sci. 25 (1), 8-15 (2004).</li><li>Pani, B., Ong, H. L., Liu, X., Rauser, K., Ambudkar, I. S., Singh, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+rafts+determine+clustering+of+STIM1+in+endoplasmic+reticulum-plasma+membrane+junctions+and+regulation+of+store-operated+Ca2++entry+(SOCE.">Lipid rafts determine clustering of STIM1 in endoplasmic reticulum-plasma membrane junctions and regulation of store-operated Ca2+ entry (SOCE.</a> J. Biol. Chem. 283 (25), 17333-17340 (2008).</li><li>Levine, T., Rabouille, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum:+one+continuous+network+compartmentalized+by+extrinsic+cues.">Endoplasmic reticulum: one continuous network compartmentalized by extrinsic cues.</a> Curr. Opin. Cell Biol. 17 (4), 362-368 (2005).</li><li>Sano, R., Annunziata, I., Patterson, A., Moshiach, S., Gomero, E., Opferman, J., Forte, M., d'Azzo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM1-ganglioside+accumulation+at+the+mitochondria-associated+ER+membranes+links+ER+stress+to+Ca(2+)-dependent+mitochondrial+apoptosis.">GM1-ganglioside accumulation at the mitochondria-associated ER membranes links ER stress to Ca(2+)-dependent mitochondrial apoptosis.</a> Mol. Cell. 36 (3), 500-511 (2009).</li><li>Raturi, A., Simmen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Where+the+endoplasmic+reticulum+and+the+mitochondrion+tie+the+knot:+The+mitochondria-associated+membrane+(MAM).">Where the endoplasmic reticulum and the mitochondrion tie the knot: The mitochondria-associated membrane (MAM).</a> Biochim. Biophys. Acta. , (2012).</li><li>Giorgi, C., De Stefani, D., Bononi, A., Rizzuto, R., 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=Structural+and+functional+link+between+the+mitochondrial+network+and+the+endoplasmic+reticulum.">Structural and functional link between the mitochondrial network and the endoplasmic reticulum.</a> Int. J. Biochem. Cell Biol. 41 (10), 1817-1827 (2009).</li><li>d'Azzo, A., Tessitore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sano+Gangliosides+as+apoptotic+signals+in+ER+stress+response.">Sano Gangliosides as apoptotic signals in ER stress response.</a> Cell Death Differ. 13, 404-414 (2006).</li><li>Kroemer, G., Galluzzi, L., Brenner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+membrane+permeabilization+in+cell+death.">Mitochondrial membrane permeabilization in cell death.</a> Physiol. Rev. 87, 99-163 (2007).</li><li>Rizzuto, R., Brini, M., Murgia, M., Pozzan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdomains+with+high+Ca2++close+to+IP3-sensitive+channels+that+are+sensed+by+neighboring+mitochondria.">Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria.</a> Science. 262, 744-747 (1993).</li><li>Lynes, E. M., Bui, M., Yap, M. C., Benson, M. D., Schneider, B., Ellgaard, L., Berthiaume, L. G., Simmen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitoylated+TMX+and+calnexin+target+to+the+mitochondria-associated+membrane.">Palmitoylated TMX and calnexin target to the mitochondria-associated membrane.</a> EMBO J. 31 (2), 457-470 (2011).</li><li>Fujimoto, M., Hayashi, T., Su, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+cholesterol+in+the+association+of+endoplasmic+reticulum+membranes+with+mitochondria.">The role of cholesterol in the association of endoplasmic reticulum membranes with mitochondria.</a> Biochem. Biophys. Res. Commun. 417 (1), 635-639 (2012).</li><li>Grimm, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ER-mitochondria+interface:+the+social+network+of+cell+death.">The ER-mitochondria interface: the social network of cell death.</a> Biochim. Biophys. Acta. 1823 (2), 327-334 (2012).</li><li>Vance, J. 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. Protoc. 4 (11), 1582-1590 (2009).</li><li>Pizzo, P., Viola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+lipid+rafts:+structure+and+function.">Lymphocyte lipid rafts: structure and function.</a> Curr. Opin. Immunol. 15 (3), 255-260 (2003).</li></ol>

Nessuno degli autori ha conflitti di interesse da dichiarare.

I siti di contatto tra membrane intracellulari o tra organelli e la membrana plasmatica di cellule rappresentano dinamiche piattaforme di segnalazione per processi cellulari di base. La caratterizzazione accurata della loro funzione e la composizione sia in condizioni fisiologiche e patologiche richiede protocolli di purificazione affidabili e riproducibili. I metodi qui descritti sono stati specificamente ottimizzate dal nostro laboratorio per l&#39;isolamento e la purificazione dei MAMs e loro gemme corrispondenti del...

<table border="1"><tbody><tr><td> Nome del reagente / materiale </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td></td><td></td><td></td><td> REAGENTI </td></tr><tr><td></td><td></td><td></td><td> Frazionamento </td></tr><tr><td> Saccarosio </td><td> Fisher Scientific </td><td> S5-500 </td><td></td></tr><tr><td> Bicarbonato di Sodio </td><td> Sigma-Aldrich </td><td> S-5761 </td><td></td></tr><tr><td> Cloruro di magnesio, esaidrato </td><td> Fisher Scientific </td><td> BP214-500 </td><td></td></tr><tr><td> Cloruro di calcio, diidrato </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-mannitolo </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, Frazione V, shock termico, liofilizzato </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> Cloruro di sodio </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> Di sodio dodecil solfato (SDS) </td><td> Fisher Scientific </td><td> BP166-500 </td><td></td></tr><tr><td></td><td></td><td></td><td> Comune </td></tr><tr><td> Compresse inibitori della proteasi, completi EDTA-free </td><td> Roche </td><td> 11-873-580-001 </td><td></td></tr><tr><td></td><td></td><td></td><td> ATTREZZATURE </td></tr><tr><td> 2 ml Douce All-Glass tessuto Grinders </td><td> Kimble Chase </td><td> 885300-0002 </td><td></td></tr><tr><td> 15 ml in polipropilene Tubi per centrifuga coniche, BD Falcon </td><td> BD </td><td> 352097 </td><td></td></tr><tr><td> 30 ml a fondo rotondo in vetro da centrifuga Tubi </td><td> Kimble Chase </td><td> 45500-30 </td><td></td></tr><tr><td> 15 ml a fondo rotondo in vetro CentriFuge Tubi </td><td> Kimble Chase </td><td> 45500-15 </td><td></td></tr><tr><td> Tubi ultracentrifuga, Ultra-Clear, pareti sottili, mm 14x89 </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> Monouso in vetro borosilicato Pasteur Pipette, 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

Organelli intracellulari sono strutture altamente dinamiche con diversa forma e composizione, che sono sottoposti a stimoli intrinseci ed estrinseci delle cellule-specifiche. Le loro membrane sono spesso giustapposti a siti di contatto definiti, che diventano centri per lo scambio di molecole di segnalazione e dei componenti della membrana 1,2,3,4. Le inter-organelli microdomini di membrana che si formano tra il reticolo endoplasmatico (ER) ei mitocondri all&#39;apertura del IP3-sensibili Ca 2 + sono noti canali come i mitocondri associato-ER membrane o MAMS 4,5,6. La proteina / lipide composizione e le proprietà biochimiche di questi siti di contatto di membrana sono stati ampiamente studiati in particolare in relazione al loro ruolo nella regolazione intracellulare Ca 2 + 4,5,6. La ER serve come fonte principale di Ca 2 + intracellulare, e in questa capacità regola una miriade di processi cellulari a valle di Ca 2 + segnalazione, including post-traduzionale folding delle proteine ​​e proteine ​​maturation7. Mitocondri, invece, mantenere l&#39;omeostasi Ca 2 +, tamponando citosolico Ca 2 + concentrazione impedendo così l&#39;apertura di percorsi apoptotici a valle di Ca 2 + squilibrio 4,8. La natura dinamica delle MAMs li rende luoghi ideali di base per analizzare i meccanismi cellulari, tra cui Ca 2 + di segnalazione e regolazione di mitocondriale Ca 2 + sopravvivenza concentrazione, la biosintesi dei lipidi e dei trasporti, il metabolismo energetico cellulare e 4,9,10,11,12. Diversi protocolli sono stati descritti per la purificazione di questi microdomini di tessuto epatico e cellule coltivate 13,14. Assunzione di metodi precedentemente pubblicati in considerazione, abbiamo adattato un protocollo per l&#39;isolamento dei mitocondri e MAMs dal cervello di topo adulto. Per questa procedura, abbiamo aggiunto un ulteriore passaggio di purificazione, cioè un estrazione Triton X100, che enables l&#39;isolamento del glicosfingolipide arricchito microdomini (GEM), frazione dei MAMs. Queste preparazioni GEM condividere componenti proteiche diverse con zattere caveolae e lipidi, derivati ​​dalla membrana plasmatica o altre membrane intracellulari, e si propone di funzionare come la raccolta punti per il raggruppamento delle proteine ​​recettori e per interazioni proteina-proteina 4,15.

Sulla base della nostra esperienza con l&#39;utilizzo di questo protocollo possiamo tranquillamente lo consiglio per l&#39;isolamento e la purificazione di MAMs, gemme e frazioni mitocondriali di cervello di topo. La procedura indicata è altamente riproducibile e coerente. In figura 1 si mostra una immagine rappresentativa dello strato modo puro mitocondri e MAMs su un gradiente Percoll (passo 3.4). A definito, banda lattiginoso contenente purificata mitocondri (Mito-P) segrega al fondo del tubo ultrac...

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

Questa procedura illustra come isolare dal cervello di topo adulto i mitocondri associate membrane ER o MAMS e glicosfingolipidi-arricchiti frazioni microdomini di MAMs e preparati mitocondriali.

I mitocondri associati Membrane ER (MAMs) e glicosfingolipidi microdomini arricchiti (GEM): Isolamento dal cervello di topo

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

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JoVE Journal

Neuroscience

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

24.2K Views.  St Jude Children's Research Hospital. Questa procedura illustra come isolare dal cervello di topo adulto i mitocondri associate membrane ER o MAMS e glicosfingolipidi-arricchiti frazioni microdomini di MAMs e preparati mitocondriali.

Video: 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.

Isolamento e coltura di cellule endoteliali dal embrionali Forebrain

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

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Neuroscienze

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.

Isolamento dei capillari cerebrali da tessuto cerebrale umano fresco

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.

Isolamento e l'estrazione di RNA dei neuroni, macrofagi e Microglia da cervelli di Zebrafish larvale

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.

Isolamento di Microglia specifico della regione da un emisfero cerebrale di topi adulto per il sequenziamento profondo dell'RNA a singola cellula

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.

Studio pre-formato Fibril indotta accumulazione di z-sinucleina in primario embrionale Midbrain Dopamine Neuroni

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.

Dissezione e isolamento della glia murina da più regioni del sistema nervoso centrale

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.

Un modello di ictus fototrombotico arricchito di fibrina e sensibile al tPA

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

Quantificazione della modifica degli istoni nel cervello di topo mediante citometria a flusso

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.

Autore in primo piano: Miglioramenti nella ricerca sulla regolazione dell'espressione genica

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.

Quantificazione delle modificazioni post-traduzionali degli istoni globali utilizzando la citometria a flusso intranucleare nella microglia cerebrale di topo isolata

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