Bu çalışma Araştırma ve Geliştirme Dairesi, Tıbbi Araştırma Servisi, Gazi İşleri Bakanlığı tarafından desteklenmektedir. Bu çalışma bir VA Liyakat İnceleme Ödülü (MCL) ve Tennessee Sağlık Bilimleri Merkezi Multipl Skleroz Araştırma Fonu Üniversitesi tarafından finanse edildi.

1. Antikor Etiketleme (Şekil 1) <ol><li> 0.1 M NaHCO3 tampon yapın: <ul type="circle"><li> 8.4 g NaHCO 3 </li><li> 29.2 g NaCl </li><li> 1 litre H2O </li></ul></li><li> Bu çözelti (10.6 gr Na 2 CO 3, 29.2 g NaCl, 1 litrelik 2 H 0) ile 8.4 arasında bir pH değerine tampon getirin. </li><li> 70 ul anti-hnRNPA1 ve 500 ul NaHCO 3 tamponu ile 35 ul Atto550 NHS ilave edin ve oda sıcaklığında 1 saat boyunca döndürmek. Saat süren dönüş sonra bir diyalizer karışımı içine enjekte ve geceleme PBS 2 litre dialyze. </li><li> Ertesi gün, bir spin kolon (Amicon Ultra 0.5) kullanarak anti-hnRNPA1 diyalizlenmiş Atto550 NHS konsantre. </li><li> Atto550 NHS anti-hnRNPA1 antikor konsantre edilmiş etiketlenmiş sonra, nano-bırakma örnek konsantrasyonunun belirlenmesi. </li></ol> 2. Antikor Transfeksiyon (Şekil 2) <ol><li> Dulbecc Tohum 10 5 hücre / 500 içine ulönce transfeksiyon için 8 sıra slayt 24 saat içinde o Modifiye Kartal Orta (DMEM/F12 +10% FBS +1% antibiyotik). Hücre konfluense en az% 70 olması gerekir. </li><li> Yirmi dört saat tohumlama sonra, bir Eppendorf tüp içine Ab-deliverin reaktifi 2 ul ekleyin. </li><li> Sonra aynı Eppendorf tüp anti-hnRNPA1 antikoru etiketli Atto550 NHS 2 mikrogram (0.5 mikrogram / ul) ekleyin ve oda sıcaklığında 10-15 dk inkübe. </li><li> İnkübasyon sırasında, medya aspire ve hücre taze DMEM ortam 394 ul ekleyin. </li></ol> Not: bir kontrol olarak hareket etmek bakir hücrelerden birinin odasına 500 ul DMEM ekleyin. <ol start="5"><li> İnkübasyondan sonra, antikor karışımı, 100 ul DMEM eklemek için aşağı yukarı ve pipetleme ile karıştırmak, ve 500 ul toplam hacim için DMEM içinde hücrelere ilave edin. </li><li> Antikor teslimat 48 saat sonra ortamı değiştirin. </li></ol> Not: Protokol FITC pozitif kontrol olarak Rigg etiketli ile yinelenmiştir. 3. Verimlilik Belirlenir Canlı ve Sabit Görüntüleme <ol><li> Görüntü hücreler görüntü transfekte Atto550 NHS etiketli antikorlar bir floresan mikroskop Cy3 filtre kullanarak 48 saat canlı. </li><li> Canlı görüntüleme tamamlandıktan sonra, medya ve düzeltme hücreleri aspire. </li><li> Ortam aspire sonra, oda sıcaklığında 15 dakika için% 0.4 paraformaldehid ile hücrelerin tedavi. Hücreler 4 x 5 dk her PBS ile yıkayın. Sonra, DAPI reaktif ve düzeltme lamel içeren montaj medya ile hücreler monte. Axiovision yazılım ile sabit hücrelerin transfeksiyon etkinliği ölçün. Axiovision yazılımı taraftan değilse, sadece 3.7 &#39;de hesaplama içine el ve fiş sonuçları ederek görüntü olayları saymak. Alternatif olarak, ImageJ yazılım Bu hesaplamalar için de kullanılabilir. </li><li> Birincisi, 20x büyütmede seçtiğiniz görüntü hücrelerinin toplam sayısını saymak için &quot;Olaylar&quot; ölçümü kullanın. </li><li> Sonraki değildi hücreleri saymak &quot;Olaylar&quot; ölçümü kullanınbaşarıyla aynı 20x büyütme görüntü antikorlar ile transfekte. </li><li> Excel tarzı biçiminde ölçülen &quot;Olaylar&quot; a erişmek için, &quot;Özellikler&quot; üzerine tıklayın &quot;Ölçüm&quot; sekmesini tıklatın ve görüntülemek için tıklatın &quot;Tablo gibi.&quot; </li><li> Son olarak, iki önceden ölçülen olayları karşılaştırarak transfeksiyon verimliliği belirlemek için aşağıdaki hesaplamayı kullanın: ((Toplam hücreleri - Untransfected Hücreleri) / Toplam Hücreler) x 100 = Transfeksiyon Verimliliği. </li></ol> 4. Temsilcisi Sonuçlar Untransfected ve canlı görüntüleri (Şekil 3A) transfekte (Şekil 3B) nöronlar göre antikor etiketleme (her resimde kırmızı veya yeşil) açıkça hücreleri (Şekil 3B, C, D) içinde gözlenen olduğunu ortaya koymaktadır. Aspirasyon sonra ve antikorlar arasında veya hücre yüzeyine yapışık çıkarmak için ortam ile yıkama, etiketlenmiş antikor yine de nöron (Şekil 3C, D) içinde mevcut bulunmaktadır.<html> Transfeksiyon verimliliği hesaplamak için başarıyla transfekte değildi hücrelere göre toplam hücreleri hesaplamak için Axiovision yazılımı kullanın. Bu değerler, toplam hücrelerin yüzdesi (Şekil 4) ve transfeksiyon etkinliğini elde etmek için bir transfeksiyon etkinliği formül girilmiştir. <img alt="Şekil 1" src="/files/ftp_upload/4154/4154fig1.jpg" /> Şekil 1. Atto 550 NHS ester ile etiket antikorlar için kullanılan prosedürleri akış şeması etiketli antikorlar konsantre için gerekli adımları izledi. <a href="http://www.jove.com/files/ftp_upload/4154/4154fig1large.jpg" target="_blank">büyük bir rakam görmek için buraya tıklayın</a> . <img alt="Şekil 2," src="/files/ftp_upload/4154/4154fig2.jpg" /> Şekil 2. Kültür canlı hücrelerin içine etiketli antikorlar transferinde kullanılan prosedürlerin Akış diyagramı.arge.jpg &quot;target =&quot; _blank &quot;&gt; büyük bir rakam görmek için buraya tıklayın. <img alt="Şekil 3" src="/files/ftp_upload/4154/4154fig3.jpg" /> Şekil 3. Transfekte nöronlar 48 saat aşağıdaki transfeksiyon görüntüleri canlı. <img alt="Şekil 4," src="/files/ftp_upload/4154/4154fig4.jpg" /> Şekil 4,. Transfeksiyon etkinliği belirlemek için sayıldı olaylar ile Resimler sabit ve durulanır.

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Dis. 186, 1514-1517 (2002).</li><li>Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+potential+link+between+autoimmunity+and+neurodegeneration+in+immune-mediated+neurological+disease.">A potential link between autoimmunity and neurodegeneration in immune-mediated neurological disease.</a> J. Neuroimmunol. 235, 56-69 (2011).</li><li>Smeenk, R. 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Neuroimmunol. 204, 140-148 (2008).</li><li>Reisinger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+transfection+of+CHO+cells+with+chemically+defined+transfection+systems+and+investigation+of+their+potential+for+transient+and+stable+transfection.">Serum-free transfection of CHO cells with chemically defined transfection systems and investigation of their potential for transient and stable transfection.</a> Cytotechnology. 60, 115-123 (2009).</li><li>Weill, C. O., Biri, S., Erbacher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+lipid-mediated+intracellular+delivery+of+antibodies+into+live+cells.">Cationic lipid-mediated intracellular delivery of antibodies into live cells.</a> Biotechniques. 44, Pvii-Pxi (2008).</li><li>Radic, M. 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Immunol. 176, 68-74 (2006).</li><li>Vincent, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+'passive+transfer'+of+paraneoplastic+stiff+person+syndrome+with+antibodies+to+an+intracellular+antigen.">Successful 'passive transfer' of paraneoplastic stiff person syndrome with antibodies to an intracellular antigen.</a> Brain. 133, 3164-3165 (2010).</li><li>Vincent, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stiff,+twitchy+or+wobbly:+are+GAD+antibodies+pathogenic.">Stiff, twitchy or wobbly: are GAD antibodies pathogenic.</a> Brain. 131 (Pt 10), 2536-2537 (2008).</li><li>Magrys, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+anti-alpha-enolase+autoantibodies+in+pathogenicity+of+autoimmune-mediated+retinopathy.">The role of anti-alpha-enolase autoantibodies in pathogenicity of autoimmune-mediated retinopathy.</a> J. 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Çıkar çatışması ilan etti.

Özel moleküller ve bunların ekspresyon hipotezi test etmek için, genler ve diğer vektörler genel olarak hücre içine transfekte edilir. Bu işlemin özgün yönü kolaylıkla hedef hücre içine transfekte etmek için antikorlar yeteneğidir. Biz beş ayrı deneylerde çiftleri her bu yöntemi uygulandı. Transfeksiyon verimliliği sonuçları tüm deneyler arasında benzer olarak bulundu. Önemli olarak, yöntem, antikor yapısı veya fonksiyonu değiştirmeden hücre içine antikorların giriş sağlar. Ayrıca...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog Numarası </td></tr><tr><td> Floresan Mikroskop AxioObserver A1 </td><td> Zeiss </td><td></td></tr><tr><td> Axiovision sürüm 4.2 </td><td> Zeiss </td><td></td></tr><tr><td> Anti-rhnRNPA1 </td><td> Abcam </td><td> Ab4791 </td></tr><tr><td> FITC etiketli anti-Rigg </td><td> OZ Biosciences </td><td> AI20100 </td></tr><tr><td> Atto550 NHS ester </td><td> Sigma </td><td> 92835 </td></tr><tr><td> Ab-deliverin </td><td> OZ Biosciences </td><td> AI20100 </td></tr><tr><td> DMEM/F12 +10% FBS +1% antibiyotikler </td><td> Gibco </td><td> 11320-033 </td></tr><tr><td> Poli-D-Lizin 8 oyuklu kültür Slaytlar </td><td> BD </td><td> 354632 </td></tr><tr><td> Çok Amaçlı Döndürücü </td><td> Bilimsel Industries A.Ş. </td><td> Model 151 </td></tr><tr><td> DAPI Montaj Medya </td><td> Millipore </td><td> S7113 </td></tr><tr><td> PBS </td><td> Ambion </td><td> 9626 </td></tr><tr><td> Diyalizer </td><td> Thermo Fisher Scientific </td><td> 66380 </td></tr><tr><td> Amicon Ultra-0.5 </td><td> Millipore </td><td> UFC501096 </td></tr><tr><td> Paraformaldehit (% 4 çözelti) </td><td> Elektron Mikroskopi Bilimleri </td><td></td></tr><tr><td> Nano-Bırak </td><td> Thermo Fisher Scientific </td><td></td></tr></tbody></table>

antibody transfection into neurons as a tool to study disease pathogenesis

Antikorlar canlı sistemlerde meydana gelen çeşitli olaylar içine roman fikir edinmek için yeteneği sağlar. Hedef proteinlere yüksek oranda spesifik antikorlar üretmek için yeteneği ele alınması gereken çok hassas biyolojik soruları için izin verdi. Önemlisi, antikorlar sistemik lupus eritematozus (SLE), romatoid artrit (RA), paraneoplastik sendromlar, multipl skleroz (MS) ve insan T-lenfotropik virüsü tip 1 (HTLV-1) de dahil olmak üzere insan hastalıklarının bir dizi patogenezinde oylandı myelopati / tropikal spastik paraparezi (HAM / TSP) 1-9 ilişkilidir. Antikorları hastalığın soruşturma alanıdır ve veri antikorlar ve enflamasyon çeşitli intrasellüler moleküllerin sonuçlar arasındaki etkileşimler, hücresel mesajlaşma ve apoptoz 10 değişmiş olduğunu göstermektedir neden nasıl. MS HAM ve / TSP olan hastalarda hücre içi RNA bağlayıcı protein heterojen ribonuclear protein antikorları üretmek için gösterilmiştir A05-07 Ocak (hnRNP A1) 3, 9, 11. Son veriler içi nöronal ve yüzey antijenleri hem antikor patojenik 3, 5-9, 11 olduğunu göstermektedir. Böylece hücre içi antikor çalışması için izin veren bir prosedür: protein etkileşimleri hastalığı patogenezinde büyük fikir ödünç verirdim. Genler genellikle birincil hücreler ve kültür hücre hatları transfekte edilir, hücre içine antikorların Ancak transfeksiyon antikor yapısı veya kötü transfeksiyon verimliliği 12 değiştirilmesi engellendi. Transfeksiyondan diğer yöntemler, katyonik lipozomlar (DOTAP / DOPE) ve destek polyethylenimines (PEI) göre antikor transfeksiyonu içerir; kontroller ile karşılaştırıldığında, 12 antikor transfeksiyon içinde bir on-kat azalmaya yol, her ikisi de. Bu çalışmada gerçekleştirilen yöntem katyonik lipid-aracılı yöntemler ile benzerdir ve yoluyla antikorları ile kovalent olmayan kompleks oluşturmak için bir lipid bazlı mekanizmasını kullanır elektrostatik ve hidrophobic etkileşimleri 13. Biz non-kovalent elektrostatik ve hidrofobik etkileşimler yoluyla antikor yakalama ve hücrelerin içinde onları sunma yeteneğine bir lipid formülasyonu Ab-deliverin reaktifi kullanılmıştır. Böylece kimyasal ve genetik bağlantıları canlı hücrelerin içine fonksiyonel antikorlar teslimatı için gerekli değildir. Protein etkileşimleri 9: Bu yöntem, bize çeşitli antikor proteini yerelleştirme deneyler, hem de hücre içi antikor moleküler sonuçlarının analiz izleme ve gerçekleştirmek için sağladı. Bu protokol, biz hızla çoğaltıcı ve transfeksiyon verimliliği yüksek derecede nöron içine antikorlar transfekte nasıl gösterecektir. Örnek olarak, anti-hnRNP A1 ve anti-IgG antikorları kullanır. Transfeksiyon etkililiği, kolay ölçülmesi için biz Atto-550-NHS ve FITC-etiketli IgG ile etiketli anti-hnRNP A1 antikorları kullanılmıştır. Atto550 NHS yüksek moleküler absorbsiyon ve kuantum yi yeni bir etiketsaha. Uyarma kaynağı ve Atto550 için floresan filtreleri Cy3 (Örn. 556 Em. 578) benzer. Buna ek olarak, yüksek Atto550 fotostabilite sahiptir. FITC-etiketli IgG bu yöntem bağımlı boya çok yönlü ve olmadığını göstermek için bir kontrol olarak kullanıldı. Bu yaklaşım ve oluşturulan verilerin hedef antijenleri hücre içi antikorların insan hastalıklarının patogenezinde rolü olabileceği rolü anlaşılmasına yardımcı olacaktır.

Watch this Scientific Journal Video about Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis at JoVE.com

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Etkileşimleri ve hücre içi bir ortam içinde antikorların varlığı ile ilgili moleküler mekanizmalar üzerinde etkisi incelemek için hızlı bir yaklaşım tarif edilmektedir. Yöntem, bir lipid formülasyonu dayanan bir kovalent olmayan kompleks oluşumu kullanılarak canlı hücreler içine transfekte antikor içerir. Bu teknik, ölümsüzleştirilmiş hücre kültürleri ve primer hücreler için uyarlanabilir.

Hastalık Patogenez Eğitim için Bir Araç Olarak Nöronlar içine Antikor Transfeksiyon

Antibodies provide the ability to gain novel insight into various events taking place in living systems.  The ability to produce highly specific ...

antibody-transfection-into-neurons-as-tool-to-study-disease

Research

JoVE Journal

Neuroscience

10.7K Görüntüleme.  Veterans Administration Medical Center, Memphis, TN. Etkileşimleri ve hücre içi bir ortam içinde antikorların varlığı ile ilgili moleküler mekanizmalar üzerinde etkisi incelemek için hızlı bir yaklaşım tarif edilmektedir. Yöntem, bir lipid formülasyonu dayanan bir kovalent olmayan kompleks oluşumu kullanılarak canlı hücreler içine transfekte antikor içerir. Bu teknik, ölümsüzleştirilmiş hücre kültürleri ve primer hücreler için uyarlanabilir.

Video: Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

Lazer Yakalama Mikrodisseksiyon Drosophila Periferik Nöronlar

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Nörobilim

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent1,2. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue.
Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions3,4. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling3-8. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation7,8. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance.
Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading9-12, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas7, 8,13, and can be used to study coupling between other retinal neurons, as described here.

An easy and convenient method to determine the extent of gap junction tracer coupling between retinal neurons is described. This technique enables one to investigate the function of the electrical synapses between neurons in the intact retina under different illumination conditions and at different times of the day and night.

Cut-yükleme: Retina Nöronlar arasında Gap Junction Tracer Kaplin Kapsamı incelenmesi için yararlı bir araçtır

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Primer hipokampal nöronlar kalsiyum fosfat transfeksiyonu

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Bir Nöronal Hücre Line Neurotoxicity ve nöro ilgilendim Moleküler Pathways Delineate bir aracı olarak Real-Time Empedans tabanlı Cell Analyzer

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

Bir Model Olarak Koku Sistemi Akson Büyüme Modelleri ve Morfoloji Eğitim için<em&gt; In Vivo</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the development of several animal models of these neuromuscular disorders has provided the scientific community with different therapeutic scenarios aimed at delaying or reversing the progression of these conditions. By taking advantage of the retrograde machinery of neurons, one of these approaches has been to target skeletal muscles in order to shuttle therapeutic genes into corresponding spinal cord motor neurons. Although once promising, the success of such gene delivery approach has been hampered by the sub-optimal number of transduced motor neurons it has so far shown to yield. Motor end plates (MEPs) are highly specialized regions on the skeletal musculature that are in direct synaptic contact to the spinal cord &#945; motor neurons. In this regard, it is important to note that, so far, the efforts to retrogradely transfer genes into motor neurons were made without reference to the location of the MEP region in the targeted muscles. Here, we describe a simple protocol 1) to reveal the exact location of the MEPs on the surface of skeletal muscles and 2) to use this information to guide the intramuscular delivery and subsequent optimal retrograde transport of retrograde tracers into motor neurons. We hope to utilize the results from these tracing experiments in further studies into investigating retrograde transport of therapeutic genes to spinal cord motor neurons through the targeting of MEPs.

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

Motor End Plates boyunca İntramüsküler Enjeksiyonlar: Doğrudan Motor Nöronlar içine Servis Tarayıcıları Bir Minimal İnvaziv Yaklaşım

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

Çizgili Deneyi Disosiye Hipokampal Nöronlar kullanarak Protein Substrat çekici veya itici Aktivite Eğitim için

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Organotypic Hipokampal dilim kültürler çalışma Neuroprotection ve tümör hücrelerinin Invasiveness Model olarak

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

Microsomes ilişkili Alpha-synuclein toplamları için birincil nöronlar eksojen yönetim olarak güçlü hücre modeli liflerinde oluşumu

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This &#34;prion-like&#34; spreading can be interrupted by antibodies capable of binding and &#34;neutralizing&#34; extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Here we describe a cell-based assay to quantitatively assess tau uptake by microglia with the aim of creating an investigational tool to better characterize the mechanisms of action of anti-tau antibodies.

Antikor aracılıklı Tau izni Microglia tarafından eğitim için tahlil hücre tabanlı

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing ...