SS, KB ve Avrupa Komisyonu FP7 Hibe PIRG08-GA-2010-276989, NEUREX için Eğitim ve Araştırma Federal Bakanlığı (BMBF 01 EO 0803), ve / Alman Araştırma Vakfı Hibe scha 1442 için Fazit Vakfı Lisansüstü bursu tarafından desteklenir CS 3-1 yazarlar çakışan mali çıkarları var.

1. Karışık Kortikal Hücre İzolasyonu ve Kaplama Astrosit kültürleri için Karma kortikal hücre izolasyonu P4 fare yavruları P1 kullanılarak yapılabilir. Uygun astrosit yoğunluğu elde etmek için, her iki T75 doku kültürü şişesi başına 4 fare korteksi pup kullanmak gereklidir. Bu nedenle, aşağıdaki protokol miktarlar 4 fare yavru kullanarak bir hücre hazırlama için hesaplanmıştır. <ol><li> Diseksiyon işlemine başlamadan önce, kültür ortamı astrosit prewarm 30 ml (DMEM, yüksek glikoz +% 10 ısı ile inaktive edilmiş fetal bovin serumu +% 1 penisilin / streptomisin; tabloya bakınız) 37 ° C&#39;ye kadar 37, 1 saat boyunca hücre kültür dereceli su içinde 50 mg / ml &#39;lik bir konsantrasyon ° CO2 kuluçka makinesi içinde C, poli-D-lizin (PDL) 20 ml T75 ile kaplayın bir flask. </li><li> Diseksiyon işlemi için gerekli tüm reaktifleri ve malzemeleri hazırlamak. İhtiyacınız olanlar: cerrahi makas, düz ince forseps, düz meme forseps, kağıt havlu, çöp torbası,% 70 etanol birbuz üzerinde d 2 diseksiyon yemekleri (3.5 cm çapında) 2 ml HBSS her ile doldurulur. </li><li> Hafifçe tutun ve% 70 etanol ile yavru fare baş ve boyun püskürtün. Hayvan makas kullanarak hayvanlar feda edilir. </li><li> Kafatası ortaya çıkarmak için kafa derisi boyunca anterior posterior orta hat kesi, gerçekleştirin. </li><li> Boyun burun dikkatli kafatası kesin. İki ek kesikler beyne daha fazla erişim sağlamak için yapılır: İlk kesik olfaktor, kafatası tabanından kafatası kesmek için serebellumun alt diğeri anterior yapılır. </li><li> Düz uç forseps kullanılarak, kafatası kanatlar hafifçe yana doğru çevrilmiş ve beyin HBSS ile dolu birinci kesme çanak içine alınır ve yerleştirilir. Buz üzerinde yemeğin geri yerleştirin ve tüm 4 beyinleri hasat devam ediyor. </li><li> Disseksiyon Prosedürün geri kalan bir stereomikroskop altında gerçekleştirilir. Birincisi, olfaktor ve beyincik ince kullanarak kaldırılırdiseksiyon forseps (Şekil 1B). </li><li> , Korteksleri almak ince forseps ile beyin arka ucunu yakalamak için, hemisfer arasında orta hattan bir kesik gerçekleştirmek oluşturulan koru ile forseps ikinci bir dizi eklemek ve soyma uzağa dan korteksin plaka benzeri bir yapıya beyin. </li><li> Dikkatlice ince forseps (Şekil 1D &#39;) ile çekerek korteks hemisferden meninksler teşrih. Bu adım, meningeal hücreler ve fibroblastlar tarafından nihai astrosit kültürünün kirletmeyecek. HBSS ile dolu ikinci yemeğin içine hazırlanmış korteks hemisferlerin aktarın ve buz üzerine geri. Tüm 4 korteksleri buna göre devam edin. </li><li> Son olarak, keskin bıçakları (yaklaşık 4 ila 8 kez) kullanarak küçük parçalar halinde her korteks yarımkürede kesti. </li><li> Steril koşullar altında, transfer korteks bir 50 ml Falcon tüp içine parçalar ve 22.5 ml toplam hacmi ile HBSS ekleyin. </li><li> % 2.5 tripsin, karışımı 2.5 ml ekleyin ve inkübe37 su banyosunda doku ° C&#39;de 30 dakika süre ile. Her 10 dakikada sallayarak arada çevirerek karıştırın. </li><li> Pelet korteks doku parçaları için 300 xg&#39;de 5 dakika boyunca santrifüjleyin. </li><li> Dikkatle dekantasyon ile süpernatant kaldırmak. Doku pelet kaybetmemek için size mekanik bir pipet kullanarak tutabiliriz. Doku parçaları tek hücreler (20-30 defa) içine ayrılamaz oluncaya kadar bir 10 ml plastik pipet kullanılarak 10 mL astrosit kaplama orta ve şiddetli pipetleme eklenmesi ile tek bir hücre süspansiyonu içine doku ayrışır. Astrosit kaplama orta kullanarak 20 ml ses seviyesini ayarlayın. Bir hematocytometer kullanarak sayarak tek hücre içine kanıtı korteks doku ayrışma olabilir. 4 fare yavru korteks biri hazırlanması 10-15 x10 6 disosiye tek hücre boyun eğmek zorundadır. </li><li> T75 kültür şişelerinde gelen PDL aspire plakası 37 disosiye tek bir hücre süspansiyonu ve inkübe ° CO 2 inkübatör C. </li></ol> 2. Bir Enr Edinmeiched astrosit Kültür <ol><li> Bundan sonra karışık kortikal hücreleri ve tüm 3 gün sonra kaplama orta 2 gün değiştirin. </li><li> Astrositler konfluent ve bindirilirken mikroglia vardır astrosit katmanda maruz oturmak veya zaten astrosit tabakası (Şekil 2) ayrılmış olan 7 ila 8 gün sonra, mikroglia kaldırmak için orbital çalkalayıcı üzerinde 30 dakika boyunca 180 rpm&#39;de T75 çalkalanır. Süpernatant içeren mikroglia atın veya kültür isteyen ve mikroglia tetkik edersek, aşağı doğru döndürün ve kültür 28,29 için plaka. </li><li> 20 ml taze astrosit besiyeri ekleyin ve oligodendrosit prekürsör hücrelerinin (OPC) kaldırmak için 6 saat boyunca 240 rpm&#39;de şişeyi sallayarak devam. Bazı OPCs tamamen astrosit katmanından terketmez yana, OPC kirlenmesini önlemek amacıyla 1 dakika boyunca elle şiddetle sarsmaya devam ediyor. Süpernatantı atın veya kültür OPCs 29 isterseniz, aşağı ve plaka dönerler. </li><li> Kalan conflue DurulamaPBS ile iki kere nt astrosit tabaka, 37 CO2 kuluçka makinesi içinde 5 ml tripsin-EDTA ile inkübe eklemek ° C&#39;de, PBS aspire Astrositlerde dekolmanı her 5 dakikada kontrol edin ve elinizi (2-3 kez) ve palmiye karşı şişeyi vurarak astrositlerde dekolmanı zorlamak. </li><li> Astrositler kültürü şişesi ayrılmış, sonra, astrosit kültür ortamı, 5 dakika boyunca 180 xg&#39;de sıkma hücreler, 5 ml eklemek süpernatan aspire ve 40 ml taze ortam astrosit kaplama ekleyin. Bir T75 doku kültürü şişesi ilk hücre bölünmesinden sonra 6 1x10 çevresindeki hücreleri boyun eğmek zorundadır. 37 iki T75 kültür şişeleri ve inkübe Levha hücreleri ° CO 2 inkübatör C. Her 2 ila 3 gün orta değiştirin. </li><li> Ilk split astrositler sonra 12-14 gün deneme gerçekleştirmeden önce uygun hücre konsantrasyonu 24-48 saat içinde kaplanır. Bir T75 doku kültürü şişesi ikinci hücre bölünmesinden sonra 1.5-2 çevresindeki x10 6 hücre boyun eğmek zorundadır. </li></ol>

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Çıkar çatışması ilan etti.

Burada belirtilen yöntem, ilk olarak 1980 de 27 McCarthy ve De Vellis tarafından tarif kemirgen neonatal beyin gelen astrosit kültür hazırlanması dayanmaktadır. Postnatal P1 P4 fare beyin kortikal astrositlerde izolasyonu ve kültür modifiye metodu Burada sunulan, verimleri saf primer astrosit hızlı ve son derece tekrarlanabilir olduğunu. Bu teknik kolaylıkla böyle sıçan veya domuz ve böyle omurilik gibi diğer beyin bölgelerinden gibi diğer türler, gelen astrositler yalıtmak için transfe...

Astrositler merkezi sinir sistemi (MSS) bir çok bol hücre tipi vardır. İnsanda korteksin 1 nöron başına 1,4 astrositler vardır oysa nöronlara astrositlerde oranı, fareler ve sıçanların kortekste 01:03 olduğunu. Astrosit fonksiyonu ilgi son yıllarda önemli ölçüde artmıştır. Astrositlerde bir anahtar işlevi nöronlar 2,3 yapısal ve metabolik destek sağlayarak onların rolüdür. Astrositlerin Yeni keşfedilen rolleri işlevleri geniş bir yelpazesini kapsar. Bu gelişim 4-6 esnasında gelişen aksonlar ve bazı nöroblast göç rehberlik, sinaptik iletim fonksiyonları, nöral devreleri 7-9 ile sinaps gücü ve bilgi işleme, kan-beyin bariyeri roller (BBB) ​​oluşumu 10 ve bütünlük 11-13 içerir serebrovasküler tonu 14 ve düzenlenmesi. Astrositlerde bir diğer önemli özelliği yaralanma onların yanıttır. Patolojik koşullar altında, astrocytes reaktif hale gelir ve daha fazla ara filaman glial fibriler asidik protein (GFAP) ve inhibitör ekstrasellüler matriks (ECM) proteinleri 15,16 ekspresyonunu upregüle. Reaktif astrositler kondroitin sülfat proteoglikan (CSPG) ailesinin astrosit salgılanan ECM proteinleri çoğunlukla oluşan bir glial skar oluşması, sağlıklı doku hasarı sitesi ayırmak, bu önemli faktörler MSS yaralanması 15-17 sonrasında aksonal rejenerasyon inhibe ederler. Astrositler geç embriyogenez ve erken postnatal ömrü boyunca radyal glial (RG) hücrelerden kaynaklanır. Astrosit belirtim oluştu sonra astrosit habercileri son konumlarına göç, onlar terminal farklılaşma süreci başlar nerede. İn vivo olarak, astrositler bunların tipik morfoloji 18,19 ile gösterildiği gibi üç ila dört hafta doğumdan sonra olgun görünmektedir. RG hücre subpopülasyonu subventricular dilimi astrositler (tip B hücreleri) dönüştürmek. Both, RG ve B tipi hücrelerin sırasıyla, geliştirme sırasında ve yetişkin astrosit benzeri nöral kök hücreler (NSC&#39;lerde) olarak işlev. Astrositler, RG ve B tipi hücreler gibi de bu belirteçler sadece özellikle yetişkin astrositler etiketlemek için kullanılamaz belirten astrosit özgü glutamat taşıyıcısı (GLAST), beyin lipid-bağlayıcı protein (BLBP) ve GFAP ifade. Sağlıklı beyin, RG ve böyle kendini yenilemek için kapasite olarak B tipi hücreler sergi kök hücre potansiyeli bölmek değil yetişkin parankimal astrositler, aksine. Astrosit düzensizliği Alzheimer hastalığı 20,21, 22, Huntington hastalığı, Parkinson hastalığı 23, Rett sendromu, 24 ve 25 Alexander hastalığı da dahil olmak üzere, çeşitli patolojiler kapsamına dahil edilmiştir. Ayrıca, astrositler astrosit aktivasyonu ve astrositik glial skar 16,26 yol MSS tüm hakaretlerini tepki. Aşağıdaki beyin tr oluşturan astrositik glial skarauma veya spinal kord yaralanması nöronal rejenerasyon 15 engelleyen ana engel olarak düşünülmektedir. Hücrelerin saflaştırılmış popülasyonları ayırmak ve korumak için güvenilir yöntemler geliştirilmesi sinir sisteminin anlayışımıza önemli olmuştur. McCarthy ve de Vellis tarafından öncülük çalışmaları yenidoğan sıçan doku 27&#39;den astrositlerde neredeyse saf kültürleri hazırlamak için bugüne kadar araştırmacılar sağlar. Much fare kortikal astrositler izole etmek için biraz değiştirilmiş bir formu burada sunulan bu yöntemle, astrosit biyoloji öğrendi olmuştur. Vivo çalışmalar, astrositler yanı sıra klimalı ortamda elde edilen in vitro kültür açıklanan kullanarak tamamlayan, daha astrosit işlevleri içgörüler kazanmak için değerli araçlardır.

<table border="1"><tbody><tr><td> Çalışma solüsyonu Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Nihai konsantrasyon </td></tr><tr><td></td><td></td><td></td><td> Astrosit kültür besiyeri </td></tr><tr><td> DMEM, yüksek glukoz </td><td> Yaşam Teknolojileri </td><td> 31966-021 </td><td></td></tr><tr><td> Isı inaktive FBS, </td><td> Yaşam Teknolojileri </td><td> 10082-147 </td><td> Nihai konsantrasyon:% 10 </td></tr><tr><td> Penisilin-Streptomisin </td><td> Yaşam Teknolojileri </td><td> 15140-122 </td><td> Nihai konsantrasyon:% 1 </td></tr><tr><td></td><td></td><td></td><td> Beyin dokusu kazmak için Çözümestion </td></tr><tr><td> HBSS </td><td> Yaşam Teknolojileri </td><td> 14170-088 </td><td></td></tr><tr><td> % 2.5 tripsin </td><td> Yaşam Teknolojileri </td><td> 15090-046 </td><td> Nihai konsantrasyon:% 0.25 </td></tr><tr><td></td><td></td><td></td><td> Diğer </td></tr><tr><td> % 70 (hacim / hacim) etanol </td><td> Roth </td><td> 9065.2 </td><td></td></tr><tr><td> Poli-D-Lizin </td><td> Millipore </td><td> A-003-E </td><td> 50 mg / ml </td></tr><tr><td> Su </td><td> PAA </td><td> S15-012 </td><td> hücre kültürü notu </td></tr><tr><td> PBS </td><td> PAA </td><td> H15-002 </td><td> hücre kültürü notu </td></tr><tr><td> 0.05% deneyinpsin-EDTA </td><td> Yaşam Teknolojileri </td><td> 25300-062 </td><td></td></tr><tr><td> 0.45 mikron Steril filtre </td><td> Sartorius </td><td> 16555 </td><td></td></tr><tr><td> 3,5 cm petri </td><td> BD Falcon </td><td> 353001 </td><td></td></tr><tr><td> 15 ml Falcon tüp </td><td> BD Falcon </td><td> 352096 </td><td></td></tr><tr><td> 50 ml Falcon tüp </td><td> BD Falcon </td><td> 352070 </td><td></td></tr><tr><td> 75 cm 2 doku kültürü şişesi </td><td> BD Falcon </td><td> 353136 </td><td></td></tr><tr><td> Forseps, ince </td><td> Dumont </td><td> 2-1032, 2-1033 </td><td> # 3c; # 5 </td></tr><tr><td> Forseps, düz meme </td><td> KLS Martin </td><td> 12-120-11 </td><td></td></tr><tr><td> 13 cm cerrahi makas </td><td> Aesculap </td><td> BC-140-R </td><td></td></tr><tr><td> Stereomikroskopta </td><td> Leica </td><td></td><td> MZ7.5 </td></tr><tr><td> Stereomikroskopta + Kamera </td><td> Leica </td><td></td><td> MZ16F; DFC320 </td></tr><tr><td> Mikroskop + Kamera </td><td> Zeiss; Canon </td><td></td><td> Primo Vert; PowerShot A650 IS </td></tr><tr><td> Santrifüj </td><td> Eppendorf </td><td> 5805000.017 </td><td> Centrifuge5804R </td></tr><tr><td> Orbital Çalkalayıcı </td><td> Thermo Scientific </td><td> SHKE 4450-1CE </td><td> Maxq 4450 </td></tr><tr><td> Su banyosu </td><td> JULABO </td><td></td><td> SW20; 37 ° C </td></tr></tbody></table>

isolation and culture of mouse cortical astrocytes

Astrositler memeli beyin içinde bol bir hücre tipi, henüz çok moleküler ve işlevsel özellikleri öğrendik gerekmektedir. Astrosit in vitro hücre kültürü sistemlerinde detaylı olarak bu glial hücrelerin biyolojik fonksiyonları incelemek için kullanılabilir. Bu video protokol fare yavrular karışık kortikal hücrelerin izolasyonu ve kültürü ile saf astrositler edinme gösterir. Yöntem, canlı nöronların ve astrositlerin yokluğunda, oligodendrosit ve mikroglia, kültür içinde merkezi sinir sistemi üç ana glial hücre popülasyonları, ayrılmasına dayanır. Kültürün ilk günlerinde Örnek görüntüleri karışık bir hücre popülasyonunun varlığına göstermek ve astrositler konfluent haline ve mikroglia ve oligodendrosit ayrılmalıdır zaman timepoint göstermektedir. Ayrıca, saflık ve iyi kurulmuş için immünohistokimyasal boyamalar kullanarak kültürlü astrositlerde astrositik morfolojisi göstermek veYeni astrosit belirteçler nitelendirdi. Bu kültür sistemi kolaylıkla astrosit biyolojinin çeşitli yönlerini araştırmak için saf fare astrosit ve astrosit-şartlı ortam elde etmek için de kullanılabilir.

Tam fare beyin (Şekil 1A), serebellum ve olfaktor (Şekil 1B) kaldırılması gerekir izolasyonu üzerine. Korteksleri fare beyin sapı (Şekil 1C) ve bireysel korteks (Şekil 1D &#39;) arasında menenjlerin soyulmuş özenle (Şekil 1E) kaldırılır. Meninksler meningeal arter sistemi ve meningeal hücreler ve fibroblastlar tarafından nihai astrosit kültür kirlenmesine eksik kaldırma sonuçları ortada. <p class="jove_content...

Watch this Scientific Journal Video about Isolation and Culture of Mouse Cortical Astrocytes at JoVE.com

Isolation and Culture of Mouse Cortical Astrocytes

Astrositler normal beyin gelişimi ve işlevi, ve merkezi sinir sistemi onarımı için gerekli olan temel biyolojik süreçler katılan yönlü hücreler olduğu kabul edilmiştir. Burada santral sinir sistemi hücrelerinin bu büyük sınıf biyoloji eğitimi saf fare astrosit kültürleri elde etmek için hızlı bir prosedürdür.

Fare Kortikal astrositlerde İzolasyon ve Kültür

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

isolation-and-culture-of-mouse-cortical-astrocytes

Research

JoVE Journal

Neuroscience

91.0K Görüntüleme.  University of Freiburg. Astrositler normal beyin gelişimi ve işlevi, ve merkezi sinir sistemi onarımı için gerekli olan temel biyolojik süreçler katılan yönlü hücreler olduğu kabul edilmiştir. Burada santral sinir sistemi hücrelerinin bu büyük sınıf biyoloji eğitimi saf fare astrosit kültürleri elde etmek için hızlı bir prosedürdür.

Video: Isolation and Culture of Mouse Cortical Astrocytes

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Embriyonik Fare Hipokampal ve Kortikal nöronlar primer Nucleofection ve Kültür

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Nörobilim

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

Fare Embriyonik motor nöronlar lektin tabanlı İzolasyon ve Kültür

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

İlköğretim Rat Kortikal Nöronlar ve Glia bilaminar Co-kültür

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Prenatal Fareler gelen Hipokampal Nöronlar izolasyonu ve Kültürü

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

İnsan Mantar şeklinde Lezzet papilla hücrelerinin izolasyonu ve Kültürü

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Modelleme Astrositom Patogenez<em&gt; İn Vitro</em&gt; Ve<em&gt; In Vivo</em&gt; Kortikal Astrositler veya Şartlı, Genetiği fareler ile Nöral Kök Hücreler kullanma

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies indicate that the severity of brain injury appear to be sex dependent, and that the male neonates are more susceptible to the effects of HI-related brain injury, resulting in more severe neurological outcomes as compared to females with comparable brain injuries. The development of reliable methods to isolate and maintain highly enriched populations of sexed hippocampal astrocytes is essential to understand the cellular basis of sex differences in the pathological consequences of neonatal HI. In this study, we describe a method for creating sex specific hippocampal astrocyte cultures that are subjected to a model of in-vitro ischemia, oxygen-glucose deprivation, followed by reoxygenation. Subsequent reactive astrogliosis was examined by immunostaining for the Glial Fibrillary Acidic Protein (GFAP) and S100B. This method provides a useful tool to study the role of male and female hippocampal astrocytes following neonatal HI, separately.

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrocytes are one of the most important key players in the central nervous system (CNS). Here, we are reporting a practical method of sexed hippocampal astrocyte culture protocol in order to study the mechanisms underlying the astrocyte function in male and female neonate pups after in-vitro ischemia.

Fare Hipokampal Astrositler Seks Farkları sonra<em&gt; In-Vitro</em&gt; İskemi

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Fare Subcallosal Zone İzolasyon ve Yetişkin Nöral Kök Hücre Kültürü

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

Fare embriyonik kök hücre farklılaşma içine kortikal Interneuron Kara filmin tarih öncesi

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.