Biz protokol sağlamak için paha biçilmez bir yorum Potter grup üyelerine teşekkür etmek istiyorum. Bu çalışma, Amerikan Nöroloji Vakfı Akademisi CMH klinik araştırma eğitim bursu, Ulusal Genel Tıp Bilimleri Enstitüsü (NIGMS bir burs tarafından finanse edildi <a href="http://www.nigms.nih.gov/">http://www.nigms.nih.gov/</a> JDR (GM08169) ), NIH Ulusal Nörolojik Hastalıklar Enstitüsü ve hibe Stroke (NS054809), JDR için bir Ruth L. Kirschstein Ulusal Araştırma Hizmet Ödülü (NS060392), bir NSF EFRI hibe (0.836.017), Coulter Vakfı Translasyonel Araştırma Ödülü, ve JDR translasyonel araştırma bursu (NS007480).

<ol>
	<li>Potter, S. M., DeMarse, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+neural+cell+culture+for+long-term+studies.">A new approach to neural cell culture for long-term studies.</a> J Neurosci Methods. 110, 1-2 (2001).</li><li>Rambani, K., Vukasinovic, J., Glezer, A., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+thick+brain+slices:+an+interstitial+3D+microperfusion+system+for+enhanced+viability.">Culturing thick brain slices: an interstitial 3D microperfusion system for enhanced viability.</a> J Neurosci Methods. 180, 243-254 (2009).</li><li>Bakkum, D. J., Chao, Z. C., Potter, S. 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J., Cotman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+growth+of+hippocampal+neurons+in+defined+medium+at+low+density:+advantages+of+a+sandwich+culture+technique+or+low+oxygen.">Survival and growth of hippocampal neurons in defined medium at low density: advantages of a sandwich culture technique or low oxygen.</a> Brain Res. 494, 65-74 (1989).</li><li>Lelong, I. H., Petegnief, V., Rebel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+cells+mature+faster+on+polyethyleneimine+coated+plates+than+on+polylysine+coated+plates.">Neuronal cells mature faster on polyethyleneimine coated plates than on polylysine coated plates.</a> J Neurosci Res. 32, 562-568 (1992).</li><li>Potter, S. M., Pine, J., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+transplant+staining+with+DiI+and+vital+imaging+by+2-photon+laser-scanning+microscopy.">Neural transplant staining with DiI and vital imaging by 2-photon laser-scanning microscopy.</a> Scanning Microsc Suppl. 10, 189-199 (1996).</li><li>Rolston, J. D., Gross, R. E., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+low-cost+multielectrode+system+for+data+acquisition+enabling+real-time+closed-loop+processing+with+rapid+recovery+from+stimulation+artifacts.">A low-cost multielectrode system for data acquisition enabling real-time closed-loop processing with rapid recovery from stimulation artifacts.</a> Front Neuroengineering. 2, (2009).</li><li>Wagenaar, D. A., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+all-channel+stimulator+for+electrode+arrays,+with+real-time+control.">A versatile all-channel stimulator for electrode arrays, with real-time control.</a> J Neural Eng. 1, 39-45 (2004).</li><li>van Pelt, J., Wolters, P. S., Corner, M. A., Rutten, W. L., Ramakers, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+characterization+of+firing+dynamics+of+spontaneous+bursts+in+cultured+neural+networks.">Long-term characterization of firing dynamics of spontaneous bursts in cultured neural networks.</a> IEEE Trans Biomed Eng. 51, 2051-2062 (2004).</li><li>Wagenaar, D. A., Pine, J., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+extremely+rich+repertoire+of+bursting+patterns+during+the+development+of+cortical+cultures.">An extremely rich repertoire of bursting patterns during the development of cortical cultures.</a> BMC Neurosci. 7, (2006).</li><li>Wagenaar, D. A., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+multi-channel+stimulus+artifact+suppression+by+local+curve+fitting.">Real-time multi-channel stimulus artifact suppression by local curve fitting.</a> J Neurosci Methods. 120, 113-120 (2002).</li><li>Bakkum, D. J., Chao, Z. C., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+activity-dependent+plasticity+of+action+potential+propagation+delay+and+amplitude+in+cortical+networks.">Long-term activity-dependent plasticity of action potential propagation delay and amplitude in cortical networks.</a> PLoS One. 3 (5), e2088-e2088 (2008).</li><li>Wagenaar, D. A., Madhavan, R., Pine, J., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+bursting+in+cortical+cultures+with+closed-loop+multi-electrode+stimulation.">Controlling bursting in cortical cultures with closed-loop multi-electrode stimulation.</a> J Neurosci. 25 (3), 680-688 (2005).</li><li>Jimbo, Y., Tateno, T., Robinson, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+induction+of+pathway-specific+potentiation+and+depression+in+networks+of+cortical+neurons.">Simultaneous induction of pathway-specific potentiation and depression in networks of cortical neurons.</a> Biophys J. 76 (2), 670-678 (1999).</li><li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing nerve cells. , (1998).</li></ol>

CMH ifşa etmek için hiçbir ilgisi yoktur.<br /&gt; JDR ve SMP Axion Biosystems için danışmanlar ödenir.<br /&gt; Bu yazı ve video gönderme ücretleri ve video işleme ücretleri nezaketle Multi-Channel Sistemleri tarafından desteklenen edildi.

Bu protokol, kültürü ile ilgili talimatlar gösterir ve nöron ağlarının yanı sıra mikro-elektrod kayıt ve nöron ağları uyarıcı bir giriş korumak. ÇÇA kayıt daha yaygın sorunlardan bazıları da MEA sorun giderme bölümünde ele alındı. Boyunca tartışılmış ve çoğu zaman bu değişiklikler bazı deneysel tasarımları gerektirdiği özel koşullar optimize etmek için kullanıldığı gibi, protokolde zaman zaman farklılıklar vardır. Burada sunulan kayıt ve MCS preamp NeuroRighter 15 tasarlanmış özel oluşan uyarıcı sistem olmasına karşın, birden fazla nöron ağları ile bir arayüz sağlayabilir diğer sistemler vardır. Belirli bir kurulum seçimi spesifik deneysel gereksinimlerine bağlı olacaktır. Ancak bu kültürlerin ÇÇA üzerinde basit bir kayıt ve stimülasyon örnekleri sinaptik aktivite ve nöronal ağ bağlantısı ile ilgili karmaşık sorular içgörü sağlamak için kullanılabilir, bu protokolde verilmiştir. Yukarıda belirtildiği gibi, devam eden çalışmaları hücresel plastisite gibi süreçleri incelemek için bu teknolojiyi kullanan, geliştirme, eksitotoksisitesi ve nörodejenerasyon 6-10 Örneğin, bizim laboratuvarda yeni bir yayın tekrarlanabilir hedefe, gerçek zamanlı kapalı döngü stimülasyonu ile in vitro öğrenme yönetti gösterdi nöronal ağ etkinliği 3 yanıt. MEA kültürler genellikle finicky ve hassas olmasına rağmen, basitlik ve erişilebilirlik (sağlam hayvanlara kıyasla) sayesinde, bir ağ düzeyinde nöronal aktivitenin çalışmak için güçlü bir model sağlar.

Sarf Malzemeleri <ul><li> Tek kullanımlık şişe top filtresi (150ml, 0.2μm gözenek boyutu; Nalgene # 565-0020) </li><li> Embriyonik gün 18 zamanlı hamile kadın sıçan </li><li> Mikroelektrod diziler (B1, protokol açıklamayı görmek) </li><li> Mikroelektrod dizi kapakları (protokol, B3 açıklama bakınız) </li><li> Naylon hücre süzgeç (40μm, BD Falcon, # 352.340) </li><li> Polipropilen konik şişeleri (steril, 15ml) </li><li> Polistiren Petri kapları (35mm, steril ve 100x15mm) </li><li> Pipetleyin ipuçları (steril 20μl, 200μl ve 1000μl) </li><li> Serolojik pipetleri (5ml) </li></ul> Ekipman: <ul><li> Biyolojik güvenlik kabini / laminer akış kaputu </li><li> Hücre sayıcı </li><li> Santrifüj </li><li> Buz konteyner </li><li> Diseksiyon makas ve forseps (steril) </li><li> Laminer akış kaput mikroskop Diseksiyon </li><li> Taşınabilir elektrik Pipetman </li><li> Hemasitometre </li><li> Isofluorane ve gaz odasında </li><li> Işık mikroskobu </li><li> El Pipetman (P-20 (20μl), P-200 (200μl), P-1000 (1000μl)) </li><li> MEA kayıt sistemi (bkz. protokol, D1 açıklama) </li><li> Kemirgen Giyotin </li><li> Tri-gaz inkübatör (protokol, B15 açıklama bakınız) </li><li> Vakum şişesi kaputu bağlı aspiratör </li><li> Girdap </li><li> Su banyosu (35-37 ° C) </li></ul> Çözümler ve Reaktifler: BSA çözüm: % 5 BSA fosfat (Sigma A-9418) tuzlu pH 7.4 ve 0.2μm süzülmüş tamponlu. Bu çözüm, 4 ° C&#39;de birkaç ay önceden hazırlanan ve saklanan olabilir. Cep Orta: 90 ml Dulbecco&#39;nun Kullanıcı modifiye Eagle Kullanıcı orta (Irvine Bilimsel 9024), 10 ml at serumu (Gibco 16050), 1 ml sodyum piruvat (Gibco 11.360, 100 mM), 250 ul GlutaMAX (Gibco 35.050), ve insülin (final konsantrasyon birleştirin 2.5 mg / ml, Sigma I-5500) sonra 0.2 mikron filter.22 Sıcak geçmesine ve kullanmadan önce inkübatör hücre orta dengelenmesi. Diseksiyon papain çözüm hazırlamak için çözüm: 23 <table border="1"><tr><td> Diseksiyon çözüm (100ml stok) </td><td> 4, filtre 0,22 mikron ve mağaza birleştirin ° C </td><td></td></tr><tr><td> Bileşen </td><td> Cilt ml </td><td> Final Konsantrasyon (mM) </td></tr><tr><td> Çift distile su </td><td> 91,175 </td><td></td></tr><tr><td> 1 M MgCl 2 </td><td> 0,58 </td><td> 5.8 mM </td></tr><tr><td> 1 M CaCl 2 </td><td> 0,025 </td><td> 0,25 mm </td></tr><tr><td> 0,5 M Hepes </td><td> 0,32 </td><td> 1.6 mm </td></tr><tr><td> % 0.5 fenol kırmızısı </td><td> 0,2 </td><td> (% 0.001) </td></tr><tr><td> 0.1 N NaOH </td><td> 0,2 </td><td> 0.2 mM </td></tr><tr><td> 2 M Na2SO4 </td><td> 4,5 </td><td> 90 mm </td></tr><tr><td> 1 M K2SO4 </td><td> 3,0 </td><td> 30 mm </td></tr><tr><td> 0.1 M Kynurenic asit </td><td> 1,0 </td><td> 1 mM </td></tr><tr><td> 5 mM APV </td><td> 1,0 </td><td> 0.05 mm </td></tr></table> Bu çözüm, 4 ° C&#39;de birkaç ay önceden hazırlanan ve saklanan olabilir. DNAz I: Invitrogen (# 18.047.019). 50μl alikotları -80 ° C&#39;de dondurulmuş flaş ve saklanır Bir kısım hücre disosiasyon süreci başlamadan önce laminer akış kaputun içindeki çözülmüş. Hank dengeli tuz çözeltisi: Sigma (# 4891). 1X çözüm steril 0.2μm süzülmüş ve 4 saklanan ° C, deiyonize su ile hazırlanır Hazırda Bekleme çözüm: Kültür, günü hazırlayın. 98 ml L-15 orta (Invitrogan 11415) 2 ml B-27 takviyesi (50X, Invitrogen # 17504) ve 0.2 mikron filtre ile birleştirin. Laminin çözüm: Kültür, günü hazırlayın. 0.02mg/ml son bir konsantrasyon için 980 ul hücre orta laminin 20μl sulandırınız (1mg/ml, Sigma L-2020). 20 ul alikotları laminin (1mg/ml) -80 ° C&#39;de saklanır ve kullanımdan hemen önce çözülmüş. Çözülme tezgah üstünde gerçekleşmesi gereken ve su banyosu gibi hızlı çözülme bazen laminin jelleşme yol. Papain çözüm: 23 30-32 Sıcak 10 ml (üstte) diseksiyonu çözüm ° C 200 adet papain ekle (Sigma P-4762) ve 1.6 mg L-sistein (Sigma C-7880). Ul yaklaşık 14 0.1 N NaOH ile pH 7.4 &#39;e ayarlayın. Çözüm cl kadar yaklaşık 30 dakika boyunca inkübekulaklar, filtre 0.2 mikron, -80, 15 ml polipropilen steril konik şişeleri ve mağaza, 2 ml alikotları sıvı nitrojen ile flash dondurma ° C 35-37 ° C protokolü başlamadan önce bir kısım çözülme. Polietilenimin (PEI) çözüm: 13 PEI, 100 ul (% 50 w / v, Sigma P-3143), bir final konsantrasyon% 0.05 w / v için 100 ml sodyum borat tamponu (0.1 M, pH 8.4, Sigma B-9876) ile seyreltilir Steril filtre 0.2 mikron. Bu, önceden hazırlanmış ve ay süreyle 4 ° C&#39;de saklanabilir. Steril de-iyonize su: Otoklav ve kullanmadan önce oda sıcaklığına kadar soğumasını bekleyin. Tripsin (EDTA, Gibco 25200 ile% 0.25): 1ml alikotları -80 ° C&#39;de dondurulmuş flaş ve saklanır Alikot kullanılmadan önce 35-37 ° C su banyosunda çözülmüş.

how to culture, record and stimulate neuronal networks on micro-electrode arrays (meas)

Geçen yüzyıl boyunca, dünyanın pek çok Nörobilimadamları nöral ağların nasıl çalıştığını anlamak için hayatlarını adamış ve çeşitli hastalıkların neden çalışmayı durdurur. Çalışmalar nöropatolojik gözlem, genetik etiketleme ile floresan mikroskopi ve ayrışmış nöronlar ve dilim hazırlıkları hem de hücre içi kayıt dahil ettik. Bu protokol, mikro-elektrot dizileri (aynı zamanda multi-elektrot diziler, ÇÇA denir) ayrışmış nöronal kültürlerin büyüyen içeren bir başka teknoloji tartışıyor. Diğer teknolojilere kıyasla, bu sistemi kullanarak birden fazla avantajı vardır. ÇÇA üzerindeki kontrol grubuyla nöronal kültürlerin ağ aktivite dizinin birden fazla elektrotlar üzerinden elektrik stimülasyonu dizileri ile manipüle edilebilir olan basitleştirilmiş bir model sağlar. Ağ küçük olduğundan, stimülasyon etkisi gözlemlenebilir alanları, bozulmadan hazırlıklarında söz konusu değildir sınırlıdır. Çeşitli görüntüleme teknikleri ile izlemek için morfolojisi kolay değişiklik yapmadan bir tek tabaka hücreleri büyümek. Son olarak, in vitro olarak bir yıl boyunca diğer kültür teknikleri ile doğasında herhangi bir net zaman sınırlamaları kaldırır ÇÇA üzerindeki kültürleri hayatta. 1 Dünya çapında laboratuvar ve diğerleri nöron ağları hakkında önemli sorular sormak için bu teknolojiyi kullanıyor. Bu protokolün amacı, bakımını kurma gelen kayıt ve elektriksel uyarıcı ÇÇA üzerindeki kültürleri için gerekli bilgileri sağlamak için, ağ fizyolojik ilgili sorular soran ve daha iyi anlaşılmasına yol açan hücre düzeyine için bir araç sağlayan in vitro ağlarda beyin fonksiyonu ve disfonksiyonu.

Watch this Scientific Journal Video about How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs at JoVE.com

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs

Bu protokol kurmak, bakımını, kayıt ve elektriksel uyarıcı ÇÇA üzerindeki kültürleri için gerekli bilgileri sağlar.<em&gt; In vitro</em&gt; Ağlar, ağ ve hücre düzeyine beyin fonksiyonu ve disfonksiyonu daha iyi anlaşılmasına yol açan fizyolojik ilgili sorular sorarak için bir araç sağlar.

Nasıl Kültür, Kayıt ve mikro-elektrot Diziler Sinir Ağları Teşvik (ÇÇA)

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays (MEAs)

54.6K Görüntüleme.  Emory University School of Medicine. Bu protokol kurmak, bakımını, kayıt ve elektriksel uyarıcı ÇÇA üzerindeki kültürleri için gerekli bilgileri sağlar. In vitro Ağlar, ağ ve hücre düzeyine beyin fonksiyonu ve disfonksiyonu daha iyi anlaşılmasına yol açan fizyolojik ilgili sorular sorarak için bir araç sağlar.

Video: How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Nörotoksisite Yüksek İçerik Analizi Nöronal ve Astrosit Hücre Co-Kültür Testi

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biyoloji

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

Civciv Ex ovo Kültür ve Ex ovo CAM Testi: Gerçekten Nasıl Çalışır?

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Bakteriyel Fonksiyonel Ağlar ve tanımında Haritalama<em&gt; Escherichia Coli</em&gt; Sentetik Genetik Diziler kullanarak

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

Multiplexed kemostat Diziler Tasarımı ve Kullanımı

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Mutlak Protein Niceleme Kütle Spektrometresi İzleme Seçilen Tepki

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Ekstraselüler Asit Üretim Ölçüm ve Analiz Glikolitik Hızı belirleme

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Techniques to Induce and Quantify Cellular Senescence

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated &#946;-galactosidase activity (SA-&#946;-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including &#947;-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-&#946;-gal, &#947;-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

Teknikler İndüklemek ve Ölçmek Hücresel Yaşlılık etmek

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

Protein ve Mikro-RNA Modülasyonunun Adipozit İşlevi Üzerindeki Etkisini Incelemek için Bir Adiposit Hücre Kültürü Modeli

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Ca2+ geçici ve L-tipi kalsiyum akımının eşzamanlı ölçümleri için yüksek kaliteli murin atriyal ve ventriküler miyositlerin izolasyonu

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Nasıl Kültür, Kayıt ve mikro-elektrot Diziler Sinir Ağları Teşvik (ÇÇA)

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