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Diseksiyonu önce hazırlanması Cerrahi aletleri: otoklav ile tüm diseksiyon makas ve forseps sterilize Lamel temizleme: <ol><li> En az 24 saat süreyle% 33 HCl 1L çim beher tüm lamelleri (çapı 24-plaka 15mm) Soak </li><li> Kalan tüm HCl kaldırmak için arada bir sallayarak 10 dakika akan su ile yıkayın </li><li> Millipore su ile durulayın. </li><li> Suyu boşaltın </li><li> % 95-98 etanol içinde lamelleri Soak </li><li> Düz bir plaka ve hava temiz bir doku Transfer lamelleri kuru lamelleri </li><li> Alüminyum folyo ile kaplayın ve otoklav, lamelleri bir cam behere aktarın Not: lameller bu aşamada saklanabilir </li><li> Kültür plakaları tek tek lamel yerleştirin. Kaplama için hazır </li></ol> Lamel kaplama: <ol><li> PBS ve filtre sterilize (0.22 mikron) ile 100 x poli-D-lisin (PDL, -20 ° C&#39;de hacimde olarak saklanır PBS içinde% 0.5 sığır serum albumin, 10 mg / ml) stok sulandırınız </li><li> 1 x 37 2 saat PDL çözüm (~ 0.15 ml / cm 2) ° C bir kuluçka makinesi ile Coat lamelleri </li><li> PDL çözüm çıkarın ve tamamen steril GKD 2 O ve kuru lamelleri ile 3 kere yıkayın </li></ol> Not: lamelleri / plakaları her diseksiyon için yeterli Coat. PDL kaplı lamelleri 4 birkaç hafta saklanabilir ° C Medya ve çözümler Diseksiyon orta (DM): 10 mM HEPES (Invitrogan 15630) ile desteklenebilir (HBSS, Invitrogen 14175) steril buz Hank Dengeli Tuz Çözeltisi (w / o Ca 2 +, Mg 2 +) kullanın. Toplam kültür ortamı,% 2 B27, 1 x Sato, 0.5 mM sodyum piruvat, 0.75mm GlutaMAX, 60μg/ml N-asetil sistein, 5 mg / ml: DMEM/NBB27 orta DMEM / Neurobasal (vol 01:01 hacim) içerir insülin, 10nm d-Biotin ve% 1 penisilin / streptomisin. 100 ml toplam kültür ortamı yapmak için, 50 ml DMEM (w / o piruvat / glutamin, Invitrogen 11960), 50 ml Neurobasal orta (Invitrogan 211.034), 375 ul GlutaMax (100 x 35.050 Invitrogen), 5.5 mg sodyum piruvat karışımı (Sigma P2256), 2 ml B27 (Invitrogan 17504), 6.3 mg N-asetilsistein (Sigma A8199), Sato hisse senedinin 1 ml d-Biotin stok (4000 x hisse senedi, 40, 25 ul (100 x stok, aşağıya bakınız) PBS içinde mcM alikotları -20 ° C. Sigma B4639), insülin 100 ul (1000 x stok, -20 alikotları gibi saklanan 0.01N HCl 5 mg / ml ° C, Sigma 16.634) ve 1 ml penisilin olarak depolanır. / streptomisin (100 x stok, Invitrogen 15140), filtre sterilize ve 4 saklamak ° C Sato 100 x stok solüsyonu: 40 ml stok yapmak için: karışımı 40 ml, 400, 400 mg BSA (Sigma A9647), mg apo-transferrin (Sigma T2252) 10 ul (25 mg / ml etanol, progesteron gibi saklanan Neurobasal alikotları -20 ° C. Sigma P8783), 64 mg putrescine (Sigma P7505) ve sodyum selenit (30 mcM PBS alikotları olarak depolanan -20 ° C, Sigma S5261) 40 ul. -20 ° C&#39;de hacimde Sato stok solüsyonu ve mağaza sterilize filtreleme Nöron kaplama orta (PM): Neurobasal Orta,% 2 B27, 2mm Glutamin (100x stok, -20 ° C, Invitrogen 25030 saklanır alikotları), 25μM glutamik asit (100 hisse senedi, -20 ° C&#39;de saklanır alikotları) ve% 1 penisilin / streptomisin. Nöron kültür ortamı (CM): Neurobasal Orta,% 2 B27, 2mm Glutamin (100x stok, -20 ° C&#39;de saklanır alikotları) ve% 1 penisilin / streptomisin. 5-FDU stok: Neurobasal orta 1mm 100x stok, 5-floro-2&#39;-dezoksiuridin (Sigma F0503) and1mM üridin (Sigma U3003). Filtre -20 ° C&#39;de sterilize ve alikot olarak depolanır Papain sindirim çözüm (diseksiyon önce taze olun): <ol><li> 4ml DM 3.2 mg L-Sistein (Sigma C-7352) çözülür </li><li> 37 1N NaOH (pH test şeritleri testi) ve su banyosu yer yaklaşık 7.4 pH ayarlama ° C Not: (aşağıya bakın) doku sindirim hemen önce bir sonraki adım gerçekleştirin. </li><li> 20 ünite / ml nihai konsantrasyonu papain </li><li> (0.22mm) sterilize Filtresi ve su banyosunda 37 ° ° C </li></ol> Tripsin inhibitörü çözüm (diseksiyon önce taze olun): <ol><li> 20ml DM 0.2 g tripsin inhibitörü (Sigma T7295) çözülür </li><li> PH kontrol edin ve 1 N NaOH ile ~ 7.4 pH ayarı </li><li> Sterilize Filtresi ve su banyosunda 37 ° ° C </li></ol> Beyin Diseksiyon Kurulum: <ul><li> Steril forseps ve makas </li><li> % 70 etanol </li><li> Temizlik kağıt mendil ve diapbankta er pedi </li><li> Bankta rahim ve fetus için buz DM 10cm Petri kapları </li><li> Diseksiyon mikroskobu Buz platformu </li><li> DM ile buz üzerinde 2-3 6cm yemekleri </li><li> 37 ° C su banyosunda sıcak PM </li></ul><ol><li> Hamile bir Kurumsal Hayvan Bakım tarafından onaylanmış bir prosedüre göre Sprague-Dawley sıçan (E16) ve İletişim Komitesi (IACUC) Euthanize </li><li> Karın bölgesi bebek bezi ped ve Spay alkol sıçan Lay </li><li> V şeklinde kesi sadece cilt kesim yapmak için karın cilt ve bir makas tutmak için cımbız kullanın </li><li> Yayıldı dışında cilt ve kas tabakası ile kesmek için başka bir steril cımbız ve makas kullanın </li><li> Embriyo keseler zincir Özü ve 10cm çanak içine buz DM içeren bir transfer </li><li> Mikrodiseksiyon makas kullanarak dikkatli bir şekilde her bir embriyo kesesi ve beyinden gelen her embriyo kaldırmak ve bir buz platformu DM temiz bir 10cm çanak içine beyinleri serbest </li><li> Laminer kaput ve bir diseksiyon mikroskobu altında, orta beyin / arka beyin bölümleri kaldırmak ve meninksler kaldırmak için ince forseps kullanımı, ve bir buz paketi DM yeni bir 6cm çanak temizlenmiş korteks / hipokampus transfer Not: Bu aşamada, doku, enzim sindirim için hazırdır . </li><li> Make papain ve tripsin inhibitörü çözüm ve sterilize </li><li> Korteks gelen DM aşırı / hippocampi çıkarın </li><li> Hazırlanan papain sindirim çözüm 4ml ekleyin </li><li> Tam olarak 5 dakika 50ml tüp tüm içerik aktarma ve bir su banyosunda 37 Falcon tüp yeri ° C </li><li> Papain çözüm pipet yardımıyla çıkarın </li><li> 2-3 dk 5ml tripsin inhibitörü çözüm, girdap tüp ve su banyosunda 37 ° ° C </li><li> Tripsin inhibitörü çözümü çıkarın </li><li> Adım 13 ve 14 üç kez tekrarlayın </li><li> 20ml sıcak PM Ekle </li><li> Tüm kümeleri ortadan kalkana kadar (~ 20-30 kez) Karışım </li><li> 7dk için 100xg santrifüjleyin </li><li> 10ml PM pelletini tekrar santrifüj tarafından iki kez yıkayın </li><li> Düzenli nöron kültürlere veya toplu kültürler için NBB27/DMEM 10ml PM pelletini tekrar </li><li> 70μm bir hücre eleklerden hücrelerin Pass </li><li> Canlı hücre sayımı </li><li> Hücreleri normal nöron kültürler için aşağıda açıklanan veya levha olarak toplam kültürler geçin Not: Bu aşamada, yüksek oranda zenginleştirilmiş nöron kültürler deney amacına bağlı olarak yoğunluğu 200-640 hücre / mm 2, PDL kaplama plakalar üzerine ayrışmış hücreleri kaplama ile elde edilebilir. Hücreleri (~ 2-4h) bağlı sonra, sıcak PM orta yerine. 4 gün uzun süre, kültür varsa, sıcak CM ile orta yarısı değiştirin. 5-FDU (son konsantrasyon 10 mcM) nöronal kültürlerin, mitoz inhibitörü yüksek derecede saflaştırılmış için (DIV2 3 sırasında yani), non-nöronal hücre proliferasyonunu inhibe etmek DIV2 eklendi olabilir. 2 gün boyunca CM kurtarma sonra hücreler darbe tam bir orta değişikliğinin ardından başka bir 2 gün (DIV 6-7) 5-FDU ile yeniden tedavi edilir. Bundan sonra, taze, sıcak CM, her 3-4 günde bir orta yarısı değiştirin. 4 hafta kadar Nöronlar geçerli değildir. </li></ol> Agregalar hazırlık ve kaplama <ol><li> 2x10 6 hücre / ml yoğunlukta 1x Sato, 10 ng CNTF ve 10μM forskolin NBB27/DMEM orta ayrışmış hücreleri içeren Askıya Alma </li><li> Kaplanmamış 6-plaka her kuyuya hücre süspansiyonu 2ml transfer </li><li> 3 gece Kültür. Her gün, hafifçe bir kez P1000 Pipetman kullanarak hücrelerin tekrar süspansiyon Not: Hücreler, agrega (Şekil 1) oluşturmaya başlar . </li><li> Toplam kaplama bir gün önce, mont plaka / Matrigel ile lamelleri: <ul><li> Stok Matrigel buz soğuk DMEM ile 01:20 (düşük büyüme faktörü, BD Biosciences 354.230) sulandırınız Not: Matrigel alikotları üretici protokol başına hazırlıklı olmalıdır. Tüm ipuçları, Eppendorf tüpleri ve stok hacimde yapmak için çözümler Matrigel polimerizasyonu önlemek için soğuk olmalıdır. Matrigel alikotları -80 ° C 4 ° C&#39;de muhafaza ve çözdürülen </li><li> 37 ° C inkübatör geceleme seyreltilmiş Matrigel çözüm 300μl/well Coat önceden PDL kaplı lamelleri </li><li> Sıcak PBS ile yıkayın, daha sonra ılık steril GKD 2 O tabak yıkama ve inkübatör plakaları bırakın. </li></ul></li><li> 3 gün toplam oluşumundan sonra hafifçe 50 ml Falcon tüp içine 200μm örgü ile süspansiyon tekrar hücreleri tekrar süspansiyon ve elek. Agrega ağırlık (~ 3-5sn) tarafından tüp altına yerleşmek için izin verin </li><li> Dikkatle süpernatant kaldırmak </li><li> Orta ekle, yavaşça agrega tekrar süspansiyon haline getirin ve onları tekrar yerleşmek. Ölü hücreleri tek tek hücreler ve enkaz kaldırmak için bu işlemi birkaç kez tekrarlayın. Süpernatantı hala olmayan toplu hücreler ve debri içeren olup olmadığını kontrol etmek için mikroskop kullanın.s </li><li> Yavaşça aynı NBB27/DMEM orta agrega (Şekil 1) tekrar süspansiyon ve agrega saymak </li><li> 25-30 etrafında aggreggats/50ul agrega yoğunluğunu ayarlayın </li><li> Toplam süspansiyon kaplama için 2ml Eppendorf tüplerine transferi alikotları (500-1000μl) Not: agrega tüplerin dibine çökmemesi için bu yana, toplam süspansiyon çoklu tüpler kaplama yapmak çok önemlidir. Sık sık benzer her lamel kaplama agrega sayısı sağlamak için, kaplanmış lamelleri veya kültür plakalar üzerine onları ekim önce hafifçe agrega tekrar süspansiyon haline getirin. </li><li> Inkübatör Matrigel kaplı lamelleri içeren kültür plakasına çıkartın. Her kuyudan çözüm çıkarın lamelleri sıcak PBS ve GKD 2 O ile yıkayın ve ardından da kuru kısaca . Toplam tohumlama için artık hazır. </li><li> Bir tüp, onlara tohumlama önce eşit bir şekilde dağıtılacaktır izin agrega süspansiyon içeren tersine çevirin. Her lamel merkezi haline toplam süspansiyon 50μl yerleştirin ve hafifçe agrega lamel eşit takmak için izin başka bir rahatsızlık olmaksızın inkübatör plaka koymak Not: agrega yoğunluğu çok önemlidir. Eğer agrega numaralı seribaşı birbirine çok yakın ya da ekim yoğunluğu yüksek ise, onlar büyüdükçe birlikte birleştirme eğilimi gösterirler. </li><li> Her iyi 4-6 saat sonra ya da erken ertesi sabah ek DMEM/NBB27medium (500 ul) </li><li> Toplam kültürü korumak için her 3-4 günde bir, yarım orta değiştirmek. Orta değiştirdiğinizde, agrega bozmaya dikkatli olmalı ve bu kuyunun yan duvar boyunca orta ekleyerek hafifçe yapılır. Not: Birkaç saat plakasına bağlı agrega sonra, agrega neurite akıbet görülebilir. Aksonlar ilk iki hafta içinde hızla büyümeye ve agrega (Şekil 2) arasında bol bağlantıları oluşturur. Glial atalarıdır agregaların radyal göç ve astrositler ve olgun oligodendrosit içine zamanla ayırt. </li></ol><img src="/files/ftp_upload/1304/1304fig1.jpg" alt="Şekil 1" /> Şekil 1 yıkandıktan sonra farklı gün ve agrega agrega oluşturan hücreler Faz kontrastlı görüntüler. <img src="/files/ftp_upload/1304/1304fig2.jpg" alt="Şekil 2" /> 2 ve 12 gün Matrigel kaplı lamelleri numaralı seribaşı sonra toplu bir kültür Şekil 2. Faz kontrastlı görüntüler.

<ol>
	<li>Matthieu, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+in+rat+brain+aggregating+cell+cultures.">Myelination in rat brain aggregating cell cultures.</a> Neuroscience. 3, 565-56 (1978).</li><li>Chen, Y. i. n. g. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+rat+and+mouse+oligodendrocyte+precursor+cells.">Isolation and culture of rat and mouse oligodendrocyte precursor cells.</a> Nat. Protocols. 2, 1044-10 (2007).</li><li>Watkins, T. A., Emery, B., Mulinyawe, S., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+stages+of+myelination+regulated+by+gamma-secretase+and+astrocytes+in+a+rapidly+myelinating+CNS+coculture+system.">Distinct stages of myelination regulated by gamma-secretase and astrocytes in a rapidly myelinating CNS coculture system.</a> Neuron. 60, 555-55 (2008).</li><li>Svenningsen, A. F., Shan, W. e. i. -. S. o. n. g., Colman, D. a. v. i. d. R., Pedraza, L. i. l. i. a. n. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+method+for+culturing+embryonic+neuron-glial+cell+cocultures.">Rapid method for culturing embryonic neuron-glial cell cocultures.</a> Journal of Neuroscience Research. 72, 565-56 (2003).</li></ol>

Erken çalışmalar sinaps oluşumu ve dönme-aracılı serbest dalgalı agrega kültürler 1 olgun miyelin bildirdi. Burada açıklanan MSS agrega kültür sistemi, hücre gelişimi, göç ve in vitro farklılaşma analizi kolaylaştırmak için geleneksel 2B kültürlerin rahatlığı ile üç boyutlu agrega multipotent progenitör hücrelerin serum serbest büyüme birleştirir sistemi, sinir habercisi değiştirilebilir ve kullanılabilir hücre araştırma ve soruşturma nöron glia etkileşimle...

preparation of rat brain aggregate cultures for neuron and glia development studies

Bir in vitro sistemde, merkezi sinir sistemi olgun nöronlar ve glia (MSS) içine atalarıdır özetlediği geliştirme ve farklılaşma AXO glial etkileşimleri, özellikleri ve multipotent atalarıdır farklılaşma ve oligodendroglial ilerlemesini araştırmak Nörobilimadamları için güçlü bir platform sağlayacak hücresel ve moleküler düzeyde soy hücreleri. Biz burada, serum serbest bir ortamda 3-4 hafta kadar devam edebilir ve nöron glia etkileşim ve MSS miyelinasyonun incelemek için bir model olarak laboratuvarda kullanılan embriyonik sıçan forebrains MSS agrega kültür sistemi açıklanmaktadır. Bu video klibi, E16 sıçan beyin bu MSS agrega kültürleri izole ve büyümek nasıl göstereceğiz. Ayrıca, aynı beyin diseksiyonu, zenginleştirilmiş düzenli ayrışmış nöronal kültürlerin kolayca elde edilebilir ve çeşitli çalışmalar için kullanılan merkezi sinir sistemi nöron veya diğer hücreleri ile birlikte kültürler için kullanılır.

Watch this Scientific Journal Video about Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies at JoVE.com

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

Embriyonik fare beyin agrega kültür sistemi için protokolleri açıklanmıştır. Agrega multipotent atalarıdır geliştirmek ve nöronlar, astrositler ve oligodendrosit içine ayırt edebilirsiniz.

Nöron ve Glia Geliştirme Çalışmaları Rat Beyin Agrega Kültürler hazırlanması

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

preparation-rat-brain-aggregate-cultures-for-neuron-glia-development

Research

JoVE Journal

Biology

12.3K Görüntüleme.  Texas A&M University (TAMU). Embriyonik fare beyin agrega kültür sistemi için protokolleri açıklanmıştır. Agrega multipotent atalarıdır geliştirmek ve nöronlar, astrositler ve oligodendrosit içine ayırt edebilirsiniz.

Video: Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

Geç Stage Drosophila pupa Brain İlköğretim Nöronal Kültürler

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

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Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Nöronal Kültürler Midgastrula Sahne Drosophila Embriyolar hazırlanması

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

ALS - Motor Neuron Disease: Mechanism and Development of New Therapies

Jeffrey D. Rothstein speaks about the pathology and mechanisms underlying amyotrophic lateral sclerosis or ALS, advances in ALS research, and current strategies towards the development of therapies.

ALS Motor Nöron Hastalığı: Yeni Tedaviler ve Kalkınma Mekanizması

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

Nöron Soma ve Aksonlar compartmentalization mikroakışkan Aygıt Fabrikasyon

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

Grubuyla Fare Kortikal Nöron Kültürler hazırlanması

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

2-dGuo Boyalı Thymus Organ Kültürleri hazırlanması

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

Hazırlanması Aplysia Duyusal-motor Nöronal Hücre Kültürleri

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

Çok bölmesi MSS Nöron glia Co-kültür mikroakışkan Platformu

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

Toksin İndüksiyon ve Protein Ekstraksiyonu Fusarium Spp.</emProteomik Araştırmalar> Kültürler

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

Biyomekanik ve Mechanobiological Araştırmalar Rat Kuyruk Tendonlar hazırlanması

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...