Biz ilk protokol tasavvur eden Renata Sano katkısını kabul. Ad&#39;A. Genetik ve Gen Terapisi Sandalye Bahşedilen Çocuklar (JFC) için Kuyumcular tutar. Bu çalışma NIH hibe GM60905, DK52025 ve CA021764 ve Amerikan Lübnan Suriye İlişkili Charities (Alsaç) tarafından finanse edildi.

<html> Aşağıdaki protokol fare beyin MAM ve GEM&#39;lerle izolasyonu ve saflaştırılması için tasarlanmıştır Çözümler mitokondri, MAM ve GEM&#39;lerle izolasyonu için gerekli Fraksiyonlama ham mitokondriyal hazırlığının sağlanması: Çözelti A: 0.32 M sukroz, 1 mM NaHCO 3, 1 mM MgCl2, 0.5 mM CaCl2 + proteaz inhibitörleri (gerektiği kadar taze ekleyin) Çözüm B: 0.32 M Sakkaroz, 1 mM NaHCO 3 + proteaz inhibitörleri (gerektiği gibi, taze ekleyin) MAM İzolasyon: İzolasyon Orta: 250 mM mannitol, 5 mM HEPES pH 7.4, 0.5 mM EGTA,% 0.1 BSA Gradyan Tamponu: 225 mM mannitol, 25 mM HEPES, pH 7.5, 1 mM EGTA,% 0.1 BSA (nihai konsantrasyon) GEM&#39;lerle İzolasyon: content &quot;&gt; GEM Ekstraksiyon Tamponu: 25 mM HEPES, pH 7.5, 0.15 M NaCl,% 1 Triton X-100 + proteaz inhibitörleri (gerektiği gibi, taze ekleyin) GEM çözündürücü Tamponu: 50 mM Tris-HCI, pH 8.8, 5 mM EDTA,% 1 SDS + proteaz inhibitörleri (gerekli olduğu gibi, taze ilave ediniz). 1. İlk Adım Subselüler Fraksiyonlama: Çekirdeğin çıkarılması, Unlysed Hücreler ve Hücresel Enkaz <ol><li> CO 2 odasında fare Euthanize. </li><li> Derhal, beyin kaldırmak yarıya, buz üzerinde 2 ml tüpler yerleştirin ve tartın. </li></ol> Not: tüm prosedür boyunca beyin yarıları ayrı tutun. Aşağıda tek beyin yarı tedavisi için de geçerlidir. <ol start="3"><li> 1 ml soğuk Çözüm A. büyük bir boşluk havaneli 15 toplam vuruş ile homojen içeren önceden soğutulmuş 2 ml cam Dounce doku öğütücü koyun yarım beyin. </li><li> Buz üzerinde, 15 ml şahin tüp aktarın ve seyreltik10 hacim w / v, örneğin, 0.225 g 2,25 ml kadar homojenat. </li><li> 4. 10 dakika için 1400 x g&#39;de santrifüje örnek ° C. </li><li> Dikkatli bir şekilde buz üzerinde, süpernatanın ve bir 30 mL yuvarlak tabanlı cam santrifüj tüpüne transferi çıkarın. Pelet rahatsız değil emin olun. </li><li> Çözüm aynı 10 cilt halinde pelletini tekrar. A küçük bir açıklık havaneli, bir defada 1 ml 3-6 vuruş ile, aynı değirmeni homojenize edilir. </li><li> 4. 10 dakika ° C için 710 xg&#39;de taze 15 ml şahin tüp ve santrifüj transfer Çekirdekler ve hücre artıkları bir tablet elde edilir. </li><li> Dikkatlice adım 1.6 kaydedilen süpernatant ile süpernatant ve havuz çıkarın. </li></ol> 2. İkinci Adım: Ham Mitokondri İzolasyon <ol><li> 4, 10 dakika için 13,800 x g&#39;de santrifüjleyin süpernatan ° C. </li><li> Buz üzerinde, bir Ultra-Clear Beckman Santrifüj Tüpü supernatant aktarın </li><li> Süspanse pelet 10 ciltlik Çözüm A. Bir küçük açıklık havaneli, bir defada 1 ml 3-6 vuruş ile, aynı değirmeni homojenize edilir. </li><li> Adım 2.1 olarak santrifüjleyin. </li><li> Adımları 2.2-2.4 tekrarlayın. </li><li> Çıkan pelet zenginleştirilmiş bir mitokondrial fraksiyonu olduğunu. Havuz süpernatantlar (sitoplazmada ve ER), parafilm ile kapak ve buz üzerinde tutmak. </li><li> Küçük bir açıklık havaneli 6 vuruş ile pelet yeniden süspanse edin, 4.8 ml / g taze bir homojenleştiricinin içinde, çözelti B (g özgün beyin ağırlığı ifade edilir). </li><li> Cam Pasteur pipet kullanarak, dikkatli bir biçimde, aşağıdaki gibi tüpün altına sonradan ekleme hiçbir baloncuklar oluşur sağlanması, bir Ultra-Sil Beckman santrifüj tüpünde bir süreksiz Sakaroz Gradyan hazırlanması: Resupended Pelet (0.32 M sukroz) 3 ml 850 mM sukroz (1 mM NaHCO 3) 3 ml 1 M sukroz (1 mM NaHCO 3) 3 ml 1.2 M Sakkaroz (1 mM NaHCO içinde3) </li><li> 4 ° C&#39;de 2 saat süreyle 82,500 x g&#39;de santrifüjleyin Degrade sonunda çıkan ayrılık üç bant ve pelet üretecek: -; 2) ER, golgi, plazma membranları (0.85 M - 1 M arayüz) 0.85 M arabirimi) 1) miyelin ve diğer membran kirleticiler (0,32 M; 3) sinaptozomlar (1 M - 1.2 M arayüzü), 4) mitokondri ham (pelet) müteakip arıtma aşamaları için kullanılan </li></ol> 3. Mitokondri ilişkili ER Membran izolasyonu, MAM <ol><li> Taze eklenmiş proteaz inhibitörleri içeren 2 ml İzolasyonu Ortamda bir beyin yarısından itibaren mitokondri taze izole ham süspanse edin. </li><li> Degrade Arabellek 8 yarım beyin ml başına ve Ultra-Clear Beckman Santrifüj Tüpü yer ile% 30 Percoll degrade hazırlayın. </li></ol> * Not: d sonra doğru final konsantrasyon elde etmek için (1.43 kat) degrade tampon daha konsantre olun% 30 Percoll iluting. <ol start="3"><li> Hazırlanan degrade üstüne Katman mitokondriyal süspansiyon YAVAŞÇA kabarcıkları önlemek için. </li><li> 30 dak, 4 ° C ve 95.000 x g&#39;de santrifüje </li><li><ol class="lroman"><li> Bir cam Pasteur pipeti ile İLK Ağır Fraksiyonu (alt bant) çıkarın ve yeni bir yuvarlak tabanlı cam tüp aktarmak, buz üzerinde. </li><li> Başka bir cam Pasteur pipeti ile İKİNCİ Işık Fraksiyonu (üst bant) çıkarın ve buz üzerinde, ayrı bir taze yuvarlak alt cam tüp transfer. </li></ol></li><li> 4. 10 dakika için 6300 x g 10 ml izolasyon Orta ve santrifüj ile adım 3,5 toplanmış ve her iki fraksiyonu ° C. seyreltilir </li><li> , Adım 3.6 &#39;de elde edilen ağır fraksiyondan süpernatan atın başka bir 10 ml izolasyon Orta ile pelet tekrar süspansiyon ve adım 3.6 gibi, yeniden santrifüjlenir. Sonuçta elde edilen topak kesir saf mitokondriyal olacaktır. </li><li> Işık Frac gelen supernatant aktarıntion buz üzerinde, bir Ultra-Clear Beckman Santrifüj Tüpü adım 3.6 elde. Pelet atın. </li><li> 1 saat, 4 ° C 100.000 xg&#39;de tüp ve santrifüj doldurmak için adım 3.8 &#39;de elde edilen süpernatant yeterli İzolasyon Orta Ekle Sonuçta elde edilen topak kesir MAM olacaktır. Dikkatlice süpernatant kaldırmak ve atın. </li></ol> Not: Bu aşamada, aynı zamanda 1 saat, 4 100,000 xg&#39;de santrifüj ° C ile adım 2.6 olarak kaydedilir süpernatant. Topak kesir ER olması ve sitosolik kısmını süpernatant olacaktır. 4. Glycosphingolipid-zenginleştirilmiş Microdomains, GEM&#39;lerle çıkarımı <ol><li> Buz üzerinde 20 dakika süreyle Ekstraksiyon tampon 500 ul-1 ml saf mitokondri ve / veya MAM kesirler Lyse. </li><li> 4 de 2 dakika için 15,300 x g&#39;de santrifüjleyin lizatları ° C. </li><li> Süpernatantlar (Triton X-100 çözünür malzeme) ve yeniden santrifüj granül Collect2 dakika için çözünür malzeme kalan çıkarmak için kullanılır. (Triton X-100 Mitokondri ekstre edilmiş ve / veya Triton X-100 ekstre MAM). </li><li> Tampon solubilising yılında pelet çözünür. Bu Çözelti halindeki materyale GEM fraksiyonlar temsil eder ve daha fazla analiz için de kullanılabilir. </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>

Yazarların hiçbiri bildirmek için herhangi bir çıkar çatışması var.

Intrasellüler membranlar arasında veya organel ve hücre plazma zarı arasındaki temas siteleri temel hücresel süreçler için dinamik sinyal platformlarda temsil etmektedir. Fizyolojik ve patolojik koşullarda hem altında işlev ve kompozisyon doğru karakterizasyonu güvenilir ve tekrarlanabilir arıtma protokolü gerektirir. Burada ayrıntılı yöntemler özellikle erişkin fare beyin MAM ve bunlara karşılık gelen GEM&#39;lerle izolasyonu ve saflaştırılması için laboratuvar tarafından optimize edilmi?...

<table border="1"><tbody><tr><td> Reaktif / Malzeme Adı </td><td> Şirket </td><td> Katalog Numarası </td><td> Yorumlar </td></tr><tr><td></td><td></td><td></td><td> REAKTİFLER </td></tr><tr><td></td><td></td><td></td><td> Damıtma </td></tr><tr><td> Sakaroz </td><td> Fisher Scientific </td><td> S5-500 </td><td></td></tr><tr><td> Sodyum bikarbonat </td><td> Sigma-Aldrich </td><td> S-5761 </td><td></td></tr><tr><td> Magnezyum Klorür, hekzahidrat </td><td> Fisher Scientific </td><td> BP214-500 </td><td></td></tr><tr><td> Kalsiyum Klorür, Dihydrate </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-Manitol </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, Fraksiyon V, Isı Şok, liyofilizat </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&#39;lik </td><td></td></tr><tr><td> Sodyum klorür </td><td> Fisher Scientific </td><td> S271-3 </td><td></td></tr><tr><td> Tris Baz </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> Sodyum dodesil sülfat (SDS) </td><td> Fisher Scientific </td><td> BP166-500 </td><td></td></tr><tr><td></td><td></td><td></td><td> Ortak </td></tr><tr><td> Komple Proteaz İnhibitörleri tabletler, EDTA-ücretsiz </td><td> Roche </td><td> 11-873-580-001 </td><td></td></tr><tr><td></td><td></td><td></td><td> EKİPMAN </td></tr><tr><td> 2 ml Douce All-Cam Doku Değirmenleri </td><td> Kimble Chase </td><td> 885300-0002 </td><td></td></tr><tr><td> 15 ml Polipropilen Konik Santrifüj Tüpleri, BD Falcon </td><td> BD </td><td> 352097 </td><td></td></tr><tr><td> 30 ml Yuvarlak Dipli Cam Santrifüj Tüpleri </td><td> Kimble Chase </td><td> 45500-30 </td><td></td></tr><tr><td> 15 ml Yuvarlak Dipli Cam Centrifüg Tüpler </td><td> Kimble Chase </td><td> 45500-15 </td><td></td></tr><tr><td> Ultrasantrifüj tüpler, 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> Tek borosilikat Cam Pasteur pipetler, 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

Hücre içi organel ve hücre şekli özel akışı içsel ve dışsal uyaranlara maruz bileşim, değişen yüksek dinamik yapılardır. Onların membranlar genellikle sinyal molekülleri ve membran bileşenlerinin 1,2,3,4 alışverişi için hub haline tanımlanan temas sitelerde, yan yana. Endoplazmik retikulum (ER) ve + kanal mitokondri ilişkili-ER membran veya MAM 4,5,6 olarak bilinir IP3 duyarlı Ca 2 açılışında mitokondri arasında oluşan inter-organel zarı microdomains. Protein / lipit bileşimi ve bu membran temas sitelerin biyokimyasal özellikleri yaygın, özellikle hücre içi Ca 2 + 4,5,6 düzenlenmesinde kendi rolü ile ilgili olarak çalışılmıştır. ER +, ve bu sıfatla Ca 2 mansap hücresel süreçlerin sayısız düzenleyen hücre içi Ca 2 primer deposu olarak hizmet + sinyalizasyon dahiltranslasyon sonrası protein katlanması ve protein maturation7 uding. Mitokondri, diğer taraftan, bu şekilde alt-Ca 2 + balanssızlık 4,8 apoptotik yollarının önlenmesi başlatma sitosolik Ca2 + konsantrasyon tamponlama göre, Ca2 + homeostazını korumak. MAM ve dinamik yapısı onları ideal sitesi Ca 2 + sinyalizasyon ve mitokondrial Ca 2 + konsantrasyonu, lipit biyosentezi ve taşınması, enerji metabolizması ve hücre sağkalımı 4,9,10,11,12 düzenlenmesi de dahil olmak üzere temel hücresel mekanizmaları incelemek için yapar. Birçok protokol karaciğer doku ve kültürlenmiş hücrelerde 13,14 bu microdomains saflaştırılması için tarif edilmiştir. Dikkate önceden yayınlanmış yöntemleri alırsak, biz yetişkin fare beyin mitokondri ve MAM ve izolasyonu için bir protokol adapte var. Bu prosedür için biz ekstra bir saflaştırma adımı, yani bir Triton X100 ekstraksiyon, eklemiş hangi enables MAM arasında glycosphingolipid zenginleştirilmiş microdomain (GEM) fraksiyonu izolasyonu. Bu GEM hazırlıklar plazma zarı veya diğer intrasellüler membranların elde caveolae ve lipid sallar, birkaç protein bileşenleri paylaştığı ve reseptör proteinlerinin kümeleme için puan toplamak olarak ve protein-protein etkileşimleri 4,15 için çalışması önerilmiştir.

Biz güvenle MAM, GEM&#39;lerle ve fare beyin mitokondriyal kesirler izolasyonu ve saflaştırılması için tavsiye ederim bu protokolü kullanarak bizim deneyimlerine dayanarak. Özetlenen prosedür yüksek oranda tekrarlanabilir ve tutarlıdır. Şekil 1&#39;de bir Percoll degrade (adım 3.4) üzerine bir şekilde saf mitokondri ve MAM tabakasının temsilcisi görüntüsünü gösterebilir. MAM fraksiyonu mitokondri üzerinde yaygın ve geniş bant yukarı yapar iken içeren tanımlanmış, sütlü ...

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

Bu işlem yetişkin fare beyin mitokondri ilişkili ER membranlar veya MAM ve MAM ve mitokondriyal hazırlıkları glycosphingolipid zenginleştirilmiş microdomain fraksiyonları izole etmek için nasıl göstermektedir.

Mitokondri ilişkili ER Membranlar (MAM) ve Glycosphingolipid zenginleştirilmiştir Microdomains (GEM&#39;lerle): Fare Beyin izolasyon

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 Görüntüleme.  St Jude Children's Research Hospital. Bu işlem yetişkin fare beyin mitokondri ilişkili ER membranlar veya MAM ve MAM ve mitokondriyal hazırlıkları glycosphingolipid zenginleştirilmiş microdomain fraksiyonları izole etmek için nasıl göstermektedir.

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.

Embriyonik ön beyin gelen Endotel Hücreleri İzolasyon ve Kültürü

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

Nörobilim

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.

Yalıtım taze insan beyin dokusundan beyin kapiller

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.

Yalıtım ve RNA çıkarma larva Zebra balığı beyin sinir hücreleri, makrofajlar ve Microglia

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

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.

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.

Primer Embriyonik Fare Orta Beyin Dopamin Nöronlarında Pre-formed Fibril Indüklenen α-Sinüklein Birikiminin İncelendirilmesi

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.

Murine glia'nın Multipl Merkezi Sinir Sistemi Bölgelerinden Diseksiyonu ve İzolasyonu

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.

Fibrin ile zenginleştirilmiş ve tPA'ya duyarlı fototrombotik inme modeli

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

Akış Sitometrisi Kullanılarak Fare Beyni Histon Modifikasyonu Miktar Tayini

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.

Yazar Spotlight: Gen İfadesi Düzenleme Araştırmasındaki Gelişmeler

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.

İzole Fare Beyni Mikrogliasında İntranükleer Akış Sitometrisi Kullanılarak Global Histon Post Translasyonel Modifikasyonların Miktar Tayini

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

Yetişkin Fare Omuriliğinden Astrositlerin ve Mikroglia'nın Verimli Ekstraksiyonu

Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic cells exhibit rapid responses to environmental changes and display significant heterogeneity in terms of morphology, transcriptional profiles, and functions. While our understanding of the functions of glial cells in health and disease has advanced substantially, there remains a need for in vitro, cell-specific analyses conducted in the context of insults or injuries to comprehensively characterize distinct cell populations. Isolating cells from the adult mouse offers several advantages over cell lines or neonatal animals, as it allows for the analysis of cells under pathological conditions and at specific time points. Furthermore, focusing on spinal cord-specific isolation, excluding brain involvement, enables research into spinal cord pathologies, including experimental autoimmune encephalomyelitis, spinal cord injury, and amyotrophic lateral sclerosis. This protocol presents an efficient method for isolating astrocytes and microglia from the adult mouse spinal cord, facilitating immediate or future analysis with potential applications in functional, molecular, or proteomic downstream studies.

Yazar Spotlight: Yetişkin Fare Omuriliğinden Glial Hücrelerin İzolasyonu 

This protocol outlines the isolation of purified astrocytes and microglia from the adult mouse spinal cord, facilitating subsequent applications such as RNA analysis and cell culture. It includes detailed cell dissociation methods and procedures designed to enhance both the quality and yield of isolated cells.

İn Vitro Tahliller ve Transkriptomik Çalışmalar için Yetişkin Fare Omuriliğinden Saf Astrositlerin ve Mikroglia'nın İzolasyonu

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic ...