Name: isolation, culture and long-term maintenance of primary mesencephalic dopaminergic neurons from embryonic rodent brains
Uploaded: 2015-02-19T15:00:00.000+00:00
Duration: 8 min 45 s
Description: Watch this Scientific Journal Video about Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains at JoVE.com

This work was performed using the Division of Brain Sciences, Department of Medicine, Imperial College London startup funds to K.N.A.

NOT: hayvanlar muhafaza ve onaylanan kurumsal kurallar ve tüm hayvan prosedürlere uygun ele Imperial College&#39;ın Hayvan Refahı tarafından edildi ve Etik İnceleme Vücut (AWERB) ve Ev Ofis ve Harvard Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC), Federal ve eyalet yönetmeliklerine uygun olarak. 1. Reaktif ve Ekipmanları Kurulum <ol><li> Çözülme, kısım ve -80 ºC mağaza 1 mg / ml laminin çözümü. 1-2 ug / cm2 &#39;lik bir kaplama konsantrasyonunda elde edilen 1 ml DMEM / F12 içinde 2 ul çözülür. NOT: yavaşça buz üzerinde ve plakaları / lamelleri eklemek hemen soğuk DMEM / F12 çözülür ve çözülme laminin. </li><li> 4 ° C&#39;de 75 ml% 0.01 425 ml PBS Poly-ornitin L-ve mağaza içinde seyreltin. NOT: Çalışma çözümün raf ömrü bir ay kadardır. </li><li> Bir ye kadar 4 ° C&#39;de% 25 (ağırlık / hacim) PBS içinde BSA, pH 7.4, ve mağaza çözülürar. </li><li> (HBSS)% 50 FBS devre dışı bırakma ortamı hazırlayın. </li><li> (+) - - Glikoz (ağırlık / hacim),% 0.25 BSA (ağırlık / hacim), 50 U / ml penisilin ve streptomisin, 1 x N2 ek,% 5 (hacim / hacim) FBS,% 0.36 ilave edilmesi ile tam bir ortam hazırlama DMEM / F12 ve mağaza 4 ºC&#39;de. NOT: raf ömrü 10 gün kadardır. </li><li> En az 30 dakika boyunca% 70 (hacim / hacim) kaynayan etanol içinde yer lamelleri yağ ve otoklavın kalıntılarını gidermek için. </li><li> Talep 24 oyuklu plakalarda lamelleri ve her bir oyuğa 500 ul Poli-L-ornitin solüsyonunu ilave edin. Oda sıcaklığında doku kültürü başlık altında 1 saat inkübe ve daha sonra 500 ul su ile 3 kez yıkanır. Her plaka için 500 ul, laminin solüsyonu eklenir ve 37 ° C&#39;de, nemlendirilmiş bir doku kültürü inkübatörü içinde, O / N inkübe edin. NOT: yıkamalar 24 saat kültürden sonra hücrelerin ölümü ile poli-L-ornitin en az üç kez yıkama sonuçlarını laminin tedaviden sonra gerekli olsa da. </li><li> Doğal gaz veya meşale f üzerinde cam pipetler ipuçları parlatmaktopal. NOT: Bu adımdan sonra ucu çapı normal boyutunun yaklaşık yarısı olmalıdır. </li><li> Bir otoklav makas ve otoklav kullanarak, bir 1.5 ml mikrosantrifüj tüpleri kapakları kesilir. </li></ol> Embriyonik ventral orta beyindeki 2. Diseksiyon <ol><li> Uyuşturmak zamanlanmış-gebe (E12.5) CO 2 inhalasyon yoluyla barajlar ve daha sonra servikal dislokasyon ile euthanize. </li><li> % 70 etanol ile karın Sprey ve rahim keseler tüm maruz kadar bir karın kesi yapmak. Forseps vajinal eki kesme ve rahim kaldırmak. 100 mm Petri kabındaki, buzla soğutulmuş HBSS rahim yerleştirin. </li><li> Forseps (Şekil 1C) kullanarak, rahim ve amniyon kesesi embriyolar çıkarın. Buzla soğutulmuş HBSS yer embriyolar. NOT: Şekil 1D dikdörtgen embriyonik ventral orta beyindeki yerini gösterir. </li><li> Bir diseksiyon mikroskobu (10x büyütme) altında embriyolar yerleştirin ve mezensefalik teşrih, berzah ve mezensefalik-diensefalik sınır bölgesinde beyin kesme bioscissor ve forseps kullanarak kemer (Şekil 1E, kesi hatları için 1A Şekil bakınız). </li><li> Tüm Ortabeyin dışarı aldıktan sonra, mediodorsal orta beyin (Şekil 1F) bir kesim yapmak. </li><li> İki forseps (Şekil 1 H) kullanarak, meninksler çıkarın. NOT: KRİTİK ADIM: Bu adım, optimum verim ve nöronal hayatta kalmak için gereklidir. Kültür şartları, beyin hücreleri için uygun olduğu için, çevreleyen dokudan hücrelerin en kaplamadan sonra ölür ve bu dokudan apoptotik sinyal kültürlerinin bütünlüğüne zarar verebilir. </li><li> Bir sterilize tıraş bıçağı (Şekil 1 H, I) kullanılarak, kelebek şekli gözlemlemek ve her kanat yaklaşık yarısını kesmek için Petri kabı orta beyin doku düzeltin. NOT: Şekil 1J izole ventral Ortabeyin göstermektedir. </li><li> Transferi15 ml konik tüp buz-soğuk HBSS içine ventral orta beyindeki parça ve bir sonraki embriyo devam edin. NOT: 2,1-2,8 fazla 1 saat almamalıdır adımlar. Buz üzerinde bu zaman dilimi dışında embriyolar tutulması önemli ölçüde hücre canlılığını azaltır. </li></ol> Ventral Orta beyin hücrelerinin 3. Ayrılma <ol><li> Laminer akış kaputun altında ventral orta beyindeki parçalarını aktarın. HBSS çıkarın ve (ventral orta beyindeki kadar 12 adet yeterli hacim) konik tüp içine önceden ısıtılmış, 1 ml (37 ° C),% 0.05 tripsin-EDTA ekleyin. 5-10 dakika süreyle 37 ° C&#39;de doku inkübe edin. </li><li> Kaputun altında tripsin-EDTA çıkarın ve doku 1 ml de-aktivasyon orta (serum tripsin devre dışı bırakır) ekleyin. </li><li> Devreden Ortamı çıkarın ve iki kez tam bir ortam 1 ml doku yıkayın. Not: ayrışmış hücreleri atmadan önlemek için, boru tüm orta çıkarılması ve doku pipetleme kaçının. </li><li> 1ml tam orta ve trit ekleBir yangın cilalı cam pipet ile doku ürat. Kabarcıkların oluşumunu önlemek ve tek hücre süspansiyonu elde edilinceye kadar triturating devam edin. NOT: Genellikle 8-10 tam ayrışması için gerekli olan kısıtlı ucu geçer. </li><li> Oda sıcaklığında 5 dakika boyunca 400 x g&#39;de santrifüj hücreleri ve ortam maddesinin ayrılması. </li><li> Yeniden askıya yavaş yavaş pipetleme tam orta 1 ml pelet ve aşağı yukarı dört kez. </li></ol> 4. Kaplama Ventral Orta beyin Hücreler <ol><li> 90 ul Trypan mavi çözeltisi ile hücre süspansiyonu, 10 ul karıştırın ve bir hemositometre ile hücre sayısı. Not: Hücrelerin canlılığı, aynı zamanda, bu aşamada kontrol edilebilir. </li><li> Kapaklar üzerine her seferinde bir forseps, birini kullanarak, 100 mm Petri tabaklarda steril mikrosantrifüj tüpü kapaklar yerleştirin ve Poli-L-ornitin / laminin kaplanmış lamelleri aktarın. NOT: lamelleri yıkamayın ve bu uneve neden olabilir çünkü, laminin kurumasını önlemekN kültürleri ve hücre hayatta kalma azalma. </li><li> (Hacim harcanmadan, lamel yüzeyini kaplamak için uygun olan) tam orta eklenerek 1 ul başına 1500 hücre hacmini ayarlamak ve her bir lamel üzerine hücre süspansiyonu 100 ul (150,000 hücrelerin toplam) ekleyin. Nemlendirilmiş bir doku kültürü kuluçka makinesi içinde Petri kapları kapatın ve 1 saat süre ile inkübe (37 ° C,% 5 CO2). Not: KRİTİK ADIM: Bu adım, hücre eki ve canlılığını geliştirmek için ve her embriyo için lamelleri sayısının arttırılması için gereklidir. Seçenek olarak ise, hücreler direkt olarak oyuklara (ihtiva lamelleri), ancak hücre sayısı (yerine 150,000) oyuk başına 350,000 artırılmalıdır aktarılabilir. Doğrudan transferi nedeniyle (yanında veya lamelleri altında) plakaları takmak hücrelerin, yaklaşık 3-4 lamelleri verir iken, diğer bir deyişle, bu yöntemi kullanırken, 8-10 lamelleri, elde edilebilir. Ortalama viabiliBu yöntemi kullanarak, kültürlerin Ty, yaklaşık% 90 idi. </li></ol> 5. Kültür Büyüme ve Bakım <ol><li> Inkübasyondan 1 saat sonra, dikkatli bir şekilde 400 ul önceden ısıtılmış (37 ° C kadar) tam bir ortam ihtiva eden 24 oyuklu plakalar içinde ortam dahil olmak üzere lamelleri transferi (toplam hacim: 500 ul). 37 ºC hücreleri O / N inkübe. </li><li> Kuyular inkübasyonundan sonra, 24 saat içinde yavaşça, her bir oyuğa 500 ul komple ortam ekleyin. Not: ilk birkaç saat dopaminerjik nöronların hayatta kalma ve bu noktadan sonra önemli ölçüde değişmez hayatta kalan hücrelerin sayısı en kritik aşamadır. </li><li> İlk iki hafta için veya orta sarı olana kadar kültürlere ek ortam eklemeyin. Iki haftadan daha uzun veya taze tam önceden ısıtılmış medya (genellikle her iki hafta) sarı, medyanın değişim yarısı (500 ul) orta renk değişiklikleri için kültürleme edin. NOT: içinde dopaminerjik nöronlarkültürler bu koşullar altında daha 6 hafta boyunca hayatta olurdu. </li></ol>

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No conflict of interest declared.

Orta beyin dopaminerjik nöronları, merkezi sinir sisteminde dopamin ana kaynağıdır. Üç gruba ayrılır, substantia nigra pars compacta (SNpc), ventral tegmental alan (VTA) ve retrorubral alanı (RRF), 10, 11. SNpc ve VTA&#39;da nöronlar gibi duygu, motivasyon ve motor davranışlarının kontrolü gibi işlevleri dahil, mezokortikal mezolimbik ve nigro-striatum büyük dopaminerjik yollar, yol. SNpc ve nigrostriatal sistemi fonksiyonel bozulma nöronların ölümü, ikinci en önemli nörodejeneratif ...

Substantia nigra gelen dopaminerjik nöronların kaybı compacta Parkinson Hastalığı (PH), ikinci en yaygın nörodejeneratif bozukluk kardinal motor belirtiler neden pars. Bu mezensefalik nöronal nüfusun ölümü yatan nedeni bilinmemektedir. Kullanılmıştır geliştirilmesi ve nörofizyolojik özellikleri ve mesDA nöron, bir kaç hücre kültürü ve hayvan modeli sistemlerinde hayatta modüle edilmesi için sorumlu biyokimyasal yollar incelenmesi. Sıçan dopaminerjik hücre çizgisi 1RB 3, 27 (N27), insan dopaminerjik nöroblastoma hücre çizgisi SH-SY5Y, MN9D ve insan mezensefalik hücreleri LUHMES fare dopaminerjik melez hücre hattı olmak üzere ölümsüzleştirilmiş hücre çizgileri, biyokimyasal ve sınırlı mekanik çalışmalar 1 kullanılmaktadır -5. MesDA nöronların spesifik kaybı çalışmada, birkaç nörotoksin bazlı ve genetik modeller 6-8 geliştirilmiştir. Primer ventral orta beyin kültürlerDopaminerjik nöronlarının nöronal ve sinaptik özellikleri ve bu ortak hastalık patojenezinde rol yolları çalışmak için vazgeçilmez bir araç sağlar. Burada daha yüksek yaşama şansına sahiptir her embriyo için lamelleri verim artışı ile sonuçlanan değişiklikleri içeren mezensefalik dopaminerjik nöronların izolasyonu için detaylı bir protokol mevcut. Önceden olgun E12.5 fare mezensefalona (sıçan E14.5) kullanımı beka geliştirir. Bu yaşta nöronlar diseksiyonu sırasında sağlam hücreleri bırakır, henüz akson geliştirilen ve böylece önemli ölçüde canlılığını arttırarak stresi en aza indirir değil. Ayrıca, bu protokolün 2. bölümünde açıklandığı gibi ventral orta beyindeki dikkatli diseksiyon, daha beka geliştirir. Embriyo başına lamelleri sayısını artırmak için, alternatif bir kaplama yöntemi, bu protokolün 4. bölümde sunulmuştur. Bu altında 4 lamelleri ile karşılaştırıldığında embriyo başına en fazla 10 lamelleri bir ürün verimine yol açarStandart kaplama koşulları dolayısıyla deney başına hayvan miktarını azaltarak. Akson ve dentrites kültür sergi akıbet nöronlar, sinaptik bağlantıları oluşturmak ve canlı hücre görüntüleme, İmmünositokimya&#39;da ve elektrofizyolojik çalışmalar için bu kültürlerin uygun hale nöronal ve sinaptik belirteçlerinin varlığını ortaya koymaktadır. Ayrıca, sinir hücresi kültürlerinde kullanılması genetik ve farmakolojik manipülasyonunu daha da kolaylaştırır. In vitro olarak 2 gün itibaren dışa doğru büyümesini geliştirme çalışmaları sağlar. Ayrıca, kültür uzun dönem hayatta kalmaları (en fazla altı hafta) bu nöronların yavaş ilerleyici dejenerasyonu çalışma için uygun hale getirmektedir.

<table><tbody><tr><td>Dulbecco&#039;s modified Eagle medium nutrient mixture F-12</td><td>Invitrogen</td><td>11330</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution (HBSS) (1X), liquid</td><td>Invitrogen</td><td>24020-117</td><td></td></tr><tr><td>Fetal bovine serum, heat-inactivated (FBS)</td><td>Invitrogen</td><td>16140</td><td></td></tr><tr><td>N2 Supplement (100X), liquid</td><td>Invitrogen</td><td>17502-048</td><td></td></tr><tr><td>D-(+)-Glucose solution (45% (wt/vol) in water</td><td>Sigma</td><td>G8769</td><td></td></tr><tr><td>Bovine serum albumin BSA</td><td>Sigma</td><td>A9430</td><td></td></tr><tr><td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td><td>Sigma</td><td>L2020</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Invitrogen</td><td>15070</td><td></td></tr><tr><td>Trypsin (0.05% (wt/vol)</td><td>Invitrogen</td><td>25300</td><td></td></tr><tr><td>Bovine serum albumin (BSA) cell culture tested</td><td>Sigma&amp;nbsp;</td><td>A9418</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Sigma</td><td>P3813</td><td></td></tr><tr><td>Anti-Tyrosine hydroxylase (Th) antibody</td><td>Pel-Freez Biologicals</td><td>P60101-0&amp;nbsp;</td><td></td></tr><tr><td>Poly-L-ornithine, 0.01% solution</td><td>Sigma</td><td>P4957</td><td></td></tr><tr><td>Anti-Map2 (Microtubule associated protein-2A and -2B) antibody</td><td>Millipore</td><td>MAB3418</td><td></td></tr><tr><td>Anti-Synapsin-1 antibody</td><td>Millipore</td><td>AB1543P</td><td></td></tr><tr><td>Alexa Fluor 488 donkey anti-rabbit IgG&amp;nbsp;&amp;nbsp; antibody</td><td>Molecular Probes</td><td>A-21206</td><td></td></tr><tr><td>Alexa Fluor 594 donkey anti-rabbit IgG&amp;nbsp;&amp;nbsp; antibody</td><td>Molecular Probes</td><td>A-21207</td><td></td></tr><tr><td>Alexa Fluor 488 donkey anti-sheep IgG&amp;nbsp;&amp;nbsp; antibody</td><td>Molecular Probes</td><td>A-11015</td><td></td></tr><tr><td>Alexa Fluor 594 donkey anti-sheep IgG&amp;nbsp;&amp;nbsp; antibody</td><td>Molecular Probes</td><td>A-11016</td><td></td></tr><tr><td>Alexa Fluor 594 donkey anti-mouse IgG&amp;nbsp;&amp;nbsp; antibody</td><td>Molecular Probes</td><td>A-21203</td><td></td></tr><tr><td>Trypan blue solution (0.4% (wt/vol))</td><td>Biowhittaker</td><td>17-942E</td><td></td></tr><tr><td>Stereo Microscope</td><td>Carl Zeiss</td><td>Stemi 2000-C</td><td></td></tr><tr><td>Inverted phase contrast microscope</td><td>Carl Zeiss</td><td>Axiovert 40 C</td><td></td></tr><tr><td>Dumont Forceps</td><td>Fine Scientific Tools</td><td>May-45</td><td></td></tr><tr><td>Cover Slip Forceps &amp;ndash; Dumoxel</td><td>Fine Scientific Tools</td><td>11251-33</td><td></td></tr><tr><td>Two Dumont #45 Forceps &amp;ndash; Dumoxel</td><td>Fine Scientific Tools</td><td>11245-30</td><td></td></tr><tr><td>Blade Holder/Breaker Flat Grip - 11cm</td><td>Fine Scientific Tools</td><td>10052-11</td><td></td></tr><tr><td>Student Iris Scissors - Straight 11.5 cm</td><td>Fine Scientific Tools</td><td>91460-11</td><td></td></tr><tr><td>Fiber optic halogen illuminator</td><td>Nikon</td><td>MKII</td><td></td></tr><tr><td>Disposable Borosilicate Glass Pasteur Pipettes</td><td>Fisherbrand</td><td>13-678-20C</td><td></td></tr><tr><td>Hemocytometer</td><td>Proscitech</td><td>SVZ2NIOU</td><td></td></tr><tr><td>0.2 &amp;micro;m sterile filter units</td><td>Nalgene</td><td>NL-CE-156-4020</td><td></td></tr><tr><td>100x20 mm Petri dishes</td><td>BD Biosciences</td><td>351005</td><td></td></tr><tr><td>Round Cover Slip #1 Thickness German Glass 12 mm</td><td>Bellco Glass</td><td>1943-10012&amp;nbsp;</td><td></td></tr></tbody></table>

isolation, culture and long-term maintenance of primary mesencephalic dopaminergic neurons from embryonic rodent brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

Tirozin hidroksilaz (TH) karşı İmmünohistokimya kültürdeki hücre 0.5-1 arasında,% dopaminerjik olduğunu göstermektedir. Tirozin hidroksilaz (TH) ve mikrotübül ilişkili protein 2 (MAP2) antikorları (Şekil 3) kullanılarak, nöronal projeksiyonları kaplama sonra 2 saat içinde ortaya çıkar ve ilk gün, akson ve dendritler ayırt (Şekil 2). nöronlar fazla altı hafta boyunca hayatta ve kapsamlı akıbet göstermektedir. daha önce gösterildiği gibi kültürler nöron-...

Watch this Scientific Journal Video about Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains at JoVE.com

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Embriyonik Kemirgen Brain itibaren İlköğretim Mesensefalik dopaminerjik Nöronlar İzolasyonu, Kültür ve Uzun Vadeli Bakım

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

isolation-culture-long-term-maintenance-primary-mesencephalic

Research

JoVE Journal

Neuroscience

13.9K Görüntüleme.  Imperial College London. The causes of degeneration of midbrain dopaminergic neurons during Parkinson’s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Video: Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

Yenidoğan Rat Beyin Dokusu Karışık Glial Hücre Kültürleri Primer mikroglia İzolasyonu

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

İmmünoloji ve Enfeksiyon

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Kemirgen Yenidoğanlar İlköğretim grubuyla orta beyin dopamin Hücre Kültürleri

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Biyoloji

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 ...

Nörobilim

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

Sıçan Embriyonik Nöral Hücre İzolasyonu ve Kültür: Bir Hızlı Protokol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Fare dopaminerjik nöronlar İlköğretim Kültür

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

Hayvanlar Rapid Genotipleme Beyin Nöronlar İlköğretim Kültürler kurulması izledi

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

E14-E16 Sprague Dawley Sıçan Embriyosu İzole Karışık Primer Hipokampal ve Kortikal Nöronlardan Nörosfer Lerin Üretimi

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

Embriyonik Fare Beyincisinden İzole Edilen Nöronların Birincil Kültürü

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

Culturing and Maintaining Dopaminergic Neurons from Mouse Embryonic Brain Tissue

Source: Weinert, M. et al., <a href="https://app.jove.com/v/52475">Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains.</a> J. Vis. Exp. (2015)
This video outlines a method for cultivating dopaminergic neurons from mouse embryonic brain tissue. The process includes treating the midbrain with a proteolytic enzyme, detaching the cells, and culturing them on polymer-coated coverslips in a nutrient-rich medium with antibiotics for their growth and long-term viability.

Fare Embriyonik Beyin Dokusundan Dopaminerjik Nöronların Kültürlenmesi ve Bakımı

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Nöronal Kültür Teknikleri

Birincil Nöronal Kültür

Isolation and Culturing of Cells from Mouse Embryo Dorsolateral Telencephalons

Source: Landeira, B. S., et al. <a href="https://app.jove.com/v/56063">Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System.</a> J. Vis. Exp. (2017)
This video demonstrates a protocol for isolating and culturing primary cerebral cortex cells from the dorsolateral telencephalon of mouse embryos.