NZ sempatik nöronlar diseksiyon ve hasat onu eğitim için laboratuar üyeleri Subhas Biswas, Andrew Sproul ve Ryan Willet teşekkür etmek istiyorum. NIH-NINDS gelen bağışlarla desteklenir.

1. Diseksiyon için hazırlanması: <ol><li> Uygun kültür deneyler doğa bağlı olarak çanak (10 cm veya 6-iyi ya da 24 kuyu tabak, vb), ve sıçan kuyruklu kollajen 24 saat diseksiyonu önce ceket seçin. Sıçan kuyruğu kollajen hazırlanması ve kat kültür yemekleri kullanarak yöntemleri (1) başka bir yerde tarif edilmiştir. </li><li> % 0.25 tripsin çözüm hazırlayın fosfat tamponlu salin (PBS) (hiçbir EDTA) ve sterilize. -20 º C&#39;de 1 ml alikotları ve mağaza hazırlayın </li><li> Distile / deiyonize H 2 O. üridin, 2.5 mM ve 5-FDU içeren bir çözüm hazırlayın Filtre sterilize ve 50 ul alikotları hazırlamak. -20 º C&#39;de muhafaza </li><li> % 10 ısı inaktive at serumu (ısı 56 º C su banyosunda 30 dakika inkübe edilmesiyle inaktive) ve% 5 fetal sığır serumu: Komple Orta RPMI 1640 kültür ortamı hazırlayın. </li><li> Final Orta hazırlayın:% 1 ısı inaktive at serumu + NGF (100ηg/ml son konsantrasyon) + penisilin / streptomisin (1 / 250) + 1 / 250 uridine/5-FDU çözüm RPMI 1640 kültür ortamı (10 mcM final konsantrasyon) . Not: her zaman kullanmadan önce bu ortamda taze yapılmalıdır. 24 plaka formatında bir deney için, bir 12 ml Bu orta hazırlamanız gerekir. Bu küçük bir ekstra hazırlar tavsiye edilir. NGF (Bu protokol sonunda Tablo) çeşitli ticari kaynaklardan satın alınabilir. </li><li> Ekipman: Tek bir diseksiyon mikroskobu ve ışık kaynağına ihtiyacınız olacaktır. Odaklanarak ipuçları ile ikili Gooseneck fiber optik aydınlatma tercih edilmektedir. Iki çift, paslanmaz çelik forseps mikro diseksiyon diseksiyon makas (paslanmaz çelik bir çift: Buna ek olarak, bir (otoklavlayarak ya da en az 20 dakika boyunca% 70 etanol içinde sırılsıklam) aşağıdaki diseksiyon araçlarını temizlemek ve sterilize etmek için ihtiyacınız olacak ve Roboz her ikisi de). İki steril enjektör iğneleri (26 gague x 1 ½ inç veya benzeri) da ihtiyaç duyulacaktır. Bu bir diseksiyon tahtası, alüminyum folyo ile sarılır ve bir Kimwipe kaplı strafor 3 &quot;x3&quot; parça kurmak için gereklidir. Dekontamine% 70 etanol ile yönetim kurulu püskürtün. Son olarak, bir yangın parlatılmış cam mera pipet hazırlar. Yangın cilası için dönerken, cam pipet ve bir kaç saniye için alev ucu tutun. Inceleme üzerine, ucu pürüzsüz bir görünüm ve açılış dar olacaktır. Bu hücreler ayrılamaz adım için gerekli. Pipet steril kalması için bir hücre kültürü kaputu yangın parlatma gerçekleştirin. </li></ol> 2. Üstün Servikal Ganglia Diseksiyon: <ol><li> Yaş P0 ve P1 yenidoğan sıçan yavrularının toplayın. </li><li> Hızla kontaminasyon olasılığını azaltmak için% 70 etanol ile diseksiyon için yavru silin. </li><li> Hızla keskin bir makas çifti (bu adımı video gösterilmeyecektir) yavru başını kesmek. Kısaca aşırı kan akıtmak için steril gazlı bez karşı steril forseps kullanarak kafa tutun. </li><li> Ventral tarafı yukarı bakacak ve yönelmiştir kuyruk ucu ile diseksiyon pad üzerinde kafa yerleştirin. Kopmuş trakea kolayca görünür olmalıdır. Rağmen bir pin pad içine ağız çatı itme ve yastık içine kopmuş trakea rağmen ikinci pimi itin şu şekilde diseksiyon pad kopmuş başını sabitlemek için steril şırınga iğneleri kullanın. Diseksiyon mikroskobu altında yastık yerleştirin ve diseksiyon başlayın. </li><li> Hem forseps (her elindeki biri), trakea çevresinde temizlemek deri ve yağ kullanımı, bakımı çok derin değil. Bunu yapmak neredeyse trakeanın her iki tarafında birbirine paralel çalışan kasların bir çift gösterecektir. Not: Diseksiyon sırasında, soğuk steril PBS veya soğuk, steril, Pasteur pipeti ile teslim aşırı kan yıkayacak ve bölgenin temiz ve nemli tutmak için serum serbest RPMI 1640 orta ile sulamak gerekir. Buna bağlı olarak sadece bir veya birden fazla kez yapılabilir, ne kadar hızlı bir çalışır. Bir deneyim dissektör için, bir çift gangliyon incelemek için sadece 2-3 dakika sürer. </li><li> Bir tarafta ilk kas kesip çıkarmak için forseps kullanın. </li><li> Kas kaldırıldıktan sonra, karotis arteri görünür hale gelecektir. Genellikle, ışığın pembemsi ve kan içerir. Çevresinde görebilirsiniz diğer kan damarları daha açık renkli görünebilir ve bu nedenle karotis arteri hemen fark olmayabilir. Bu arter bir &quot;Y&quot; şeklinde bir şerit gibi göründüğünü trakea ve çatallanmasıdır yanında çalışır. Bu çatallanma önemli bir dönüm noktası olduğunu ve bulunduğu kez, SCG bulmak için nispeten kolay olacaktır. </li><li> En kaudal sonunda arter Sever ve forseps (diğer ucu hala bağlı) ile hafifçe yukarı kaldırın. Bunu yaptığınızda, thr dokusu gevşek bifurkasyonunun noktada arter bağlı ve badem şekilli, parlak görünümlü küçük bir kitle var göreceksinizöncesi ve / veya post-ganglion bağlaçlar EAD gibi. Bu üstün servikal ganglion. Öte yandan ikinci bir çift forseps yavaşça soğuk, serum serbest RPMI 1640 orta içeren küçük bir kültür çanak (35 mm) ve yer ayırmak için kullanın. </li><li> Daha sonra, aynı protokol Trakeanın diğer tarafta ganglion ayıklamak. </li></ol> 3. Ganglia Temizleme: <ol><li> Bir kez tüm ganglionlar disseke olmuştur, onlar mümkün olduğunca gereksiz doku serbest bırakılmalıdır. </li><li> Diseksiyon mikroskobu altında, karotis arter, yağ veya diğer doku parçalarıyla ganglionlar bağlı olduğunu görebilirsiniz. Bu istenmeyen maddeleri uzaklaştırmak kızdırmak için her el forseps ile çalışın. Yeri RPMI 1640 orta içeren, steril bir 15ml konik, polipropilen tüp içine ganglionlar temizlenir. </li><li> Öncesi ve / veya post-ganglion bağlaçlar da kesilmiş olmalı. Her ganglion yaklaşık 10.000 nöron vardır. Nöronlar kaplama sonra, onların neurites regrow. </li></ol> 4. Sempatik Nöron kültürü oluşturulması: <ol><li> 2 dakika boyunca 200xg ganglionlar Santrifüj ve supernatant atın. </li><li> 0,5 - 1 ml% 0.25 tripsin çözüm ganglionlar tekrar 30 dakika için 37 ˚ C su banyosunda veya inkübatör yerleştirin. Bu ganglionlar hücreleri ayrıştırmaları yardımcı olacaktır. </li><li> 30 dakika sonra, Komple Orta sulandırmak ve 2 dakika boyunca 200xg tripsin ve santrifüj nötralize etmek için 10 ml ekleyin. </li><li> Final Orta 2 ml supernatant ve tekrar süspansiyon atın. </li><li> Ayrıca yangın parlatılmış cam mera pipet ile ganglionlar yukarı ve aşağı triturating hücreleri ayrıştırmaları. Ganglionlar 20 kez Karışım ve birkaç dakika boyunca buz üzerinde tüp. </li><li> Bu arada, (yaklaşık 2 / 3, 1 / 2 mm çapında) açılış dar yapmak için daha fazla yangın lehçe pipet. Soğuyuncaya kadar birkaç dakika bekleyin ve 20 kat daha fazla ganglionlar çiğnemek. Devam ederken, orta cloudier hücrelerin tedricen ayrışmış olduğunu bildiren olacak. Kez ya da iki kez daha hücrelerin ayrıştırmaları ne kadar iyi bağlı olarak bu adımı yineleyin. Bir süre sonra, tüm ganglionlarda nöron içine ayrı ve hiç bozulmamış ganglionlar tüp içinde görünür olduğunu fark edeceksiniz. Ayrıştırmaları değil bazı doku malzeme olabilir. Bu noktada, herhangi bir undissociated enkaz altına yerleşmek böylece tüp buz oturup birkaç dakika izin verin. Şimdi dikkatli bir şekilde neredeyse tüm orta ve üst yerden başka bir tüp içinde toplamak. Undissociated enkaz toplamak etmemelerini sağlamak için tüpün alt kısmında yaklaşık 50 ul bırakın. Final orta ayrışmış hücreleri içeren tüp ekle: bu birimde kültür plakasına bir kültür nöronlar kullanarak türüne bağlıdır. Örneğin: iyi ortalama Final orta 0.5 ml 24-iyi bir yemek, iyi ortalama istihdam edilecek ise, daha sonra plakası 0.5 ganglionlar (~ 10.000 nöronlar de ortalama). 24 ganglionlar verecekti 12 yavrular, tipik bir sıçan çöp oluşur. Bu tohum 24-plaka ve Final orta toplam hacmi 12 ml yeterlidir. Not: ganglionlar kültüründe mevcut olacaktır glia ve diğer hücre türleri küçük bir nüfus da içerir. Final ortamda Uridine/5-FDU kullanılması silahların yayılmasını önlemek ve ölüm teşvik edecektir. </li><li> Yem hücreleri taze RPMI% 1 at serumu + NGF ve uridine/5-FDU ile 2 / 3 orta değiştirerek her 48 saatte bir. Not: İlk olarak, kültür, hiç neurites yuvarlak bir görünüm sempatik nöronlar içerir. Kısa neurites diseksiyonu 24 saat sonra görünür hale gelir ve kademeli olarak daha yoğun ve daha fazla zaman içinde dallı. </li></ol>

<ol>
	<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><li>Biswas, S. C., Shi, Y., Sproul, A., Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-apoptotic+Bim+Induction+in+Response+to+Nerve+Growth+Factor+Deprivation+Requires+Simultaneous+Activation+of+Three+Different+Death+Signaling+Pathways.">Pro-apoptotic Bim Induction in Response to Nerve Growth Factor Deprivation Requires Simultaneous Activation of Three Different Death Signaling Pathways.</a> The Journal of Biological Chemistry. 282 (40), 29368-29374 (2007).</li><li>Bocchini, V., Angeletti, P. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nerve+Growth+Factor:+purification+as+a+30,000-molecular-weight+protein.">The Nerve Growth Factor: purification as a 30,000-molecular-weight protein.</a> Proc. Natl. Acad. Sci. USA. (64), 787-794 (1969).</li><li>Dechant, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chat+in+the+Trophic+Web:+NGF+Activates+Ret+by+Inter-RTK+Signaling.">Chat in the Trophic Web: NGF Activates Ret by Inter-RTK Signaling.</a> Neuron. 33 (2), 156-158 (2002).</li><li>Deckwerth, T., Elliott, J. L., Knudson, C. M., Johnson, E. M., Snider, W. D., Korsmeyer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bax+is+required+for+neuronal+death+after+trophic+factor+deprivation+and+during+development.">Bax is required for neuronal death after trophic factor deprivation and during development.</a> Neuron. (17), 401-411 (1996).</li><li>Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+in+Vitro+Studies+on+the+Nerve+Growth+Factor+(NGF)+Requirement+of+Neurons+-+I.+Sympathetic+Neurons.">Quantitative in Vitro Studies on the Nerve Growth Factor (NGF) Requirement of Neurons - I. Sympathetic Neurons.</a> Developmental Biology. (58), 96-105 (1977).</li><li>Park, D. S., Morris, E. J., Padmanabhan, J., Shelanski, M. L., Geller, H. M., Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin-dependent+kinases+participate+in+death+of+neurons+evoked+by+DNA-damaging+agents.">Cyclin-dependent kinases participate in death of neurons evoked by DNA-damaging agents.</a> The Journal of Cell Biology. 143, 457-467 (1998).</li><li>Pierchala, B. A., Ahrens, R. C., Paden, A. J., Johnson, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+Growth+Factor+Promotes+the+Survival+of+Sympathetic+Neurons+through+the+Cooperative+Function+of+the+Protein+Kinase+C+and+Phosphatidylinositol+3-Kinase+Pathways.">Nerve Growth Factor Promotes the Survival of Sympathetic Neurons through the Cooperative Function of the Protein Kinase C and Phosphatidylinositol 3-Kinase Pathways.</a> The Journal of Cell Biology. 279, 27986-27993 (2004).</li></ol>

The authors have nothing to disclose.

Biz kültürlerin Sprague Dawley rat kullanın. Ganglionlar temizlik zaman ve özen. Tüm artıkları, kan damarları ve yağ çıkarın. Bu adım, daha az ya da çok homojen ve gereksiz hücre tiplerinin bir kültür sağlanmasında önemlidir. Uridine/5-FDU eklenmesi, büyük ölçüde kültür kirlenmesine kalan (mitotik) non-nöronal hücrelerin ortadan kaldırılması ya da en azından onların çoğalmasını baskıladığı. Buna ek olarak, ayrışma adım sırasında, sabırlı olun ve plakalar üzerine numaralı ser...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Forceps, Stainless steel #5</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS4976</td>
<td>&nbsp;</td>
<tr>
<td>Scissors, DEAVER straight sharp-blunt- 43mm blades - 5.5</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS6760</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640 w/ L-Glutamin</td>
<td>Reagent</td>
<td>Cellgro, Mediatech, Inc.</td>
<td>10-040-CV</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>Reagent</td>
<td>SAFC Biosciences</td>
<td>12103c</td>
<td>&nbsp;</td>
<tr>
<td>Donor Horse Serum</td>
<td>Reagent</td>
<td>JRH Biosciences</td>
<td>12449</td>
<td>Heat-inactivate by incubating in 56&deg;C waterbath for 30 minutes.</td>
</tr>
<tr>
<td>Penicillin/streptomycin</td>
<td>Reagent</td>
<td>Gibco, Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin without EDTA</td>
<td>Reagent</td>
<td>Difco</td>
<td>MT 25-050-CI</td>
<td>&nbsp;</td>
<tr>
<td>Uridine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>U3003</td>
<td>&nbsp;</td>
<tr>
<td>5-Fluoro-5' deoxyuridine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>F-8791</td>
<td>&nbsp;</td>
<tr>
<td>NGF</td>
<td>Reagent</td>
<td>Harlan Bioproduct</td>
<td>BT-5025</td>
<td>&nbsp;</td>
<tr>
<td>Dissection Microscope and light source</td>
<td>Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>15 ml polypropylene tubes</td>
<td>Tool</td>
<td>BD Falcon</td>
<td>352096</td>
<td>&nbsp;</td>
<tr>
<td>Cell culture dishes</td>
<td>Tool</td>
<td>Corning, Nunclone</td>
<td>&nbsp;</td>
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protocol for culturing sympathetic neurons from rat superior cervical ganglia (scg)

Sıçanlarda üstün servikal ganglion (SCG), sempatik nöronlar içeren küçük, parlak, badem biçimli yapılar. Bu nöronlar, baş ve boyun bölgeleri için sempatik innervations sağlamak ve iyi karakterize ve görece homojen bir nüfus (4) oluşturmaktadır. Sempatik nöronlar NGF, kullanılabilirlik, kendi kültür ve deneysel manipülasyon (2, 3, 6) kolaylaştırır, hayatta kalma, farklılaşma ve aksonal büyüme ve yaygın sinir büyüme faktörü (NGF) bağlıdır. Bu nedenlerden dolayı, kültürlü, sempatik nöronlar nöronal gelişim ve farklılaşma, ve patolojik programlanmış hücre ölümü mekanizmaları ve sinyal iletimi (1, 2, 5 ve 6) da dahil olmak üzere geniş bir yelpazede çalışmalar kullanılmaktadır. Yeni doğan sıçan ve kültür sempatik nöronlar SCG Diseksiyon çok karmaşık değildir ve oldukça hızlı bir şekilde üstesinden gelinebilir. Bu makalede, yeni doğan sıçan yavrular SCG incelemek ve sempatik nöronların kültürleri kurmak için bunları kullanmak için ne kadar detaylı bir şekilde anlatacağız. Makalede ayrıca, hazırlık adımları ve bunu başarmak için gerekli olan çeşitli reaktifler ve ekipman anlatacağız.

Watch this Scientific Journal Video about Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG at JoVE.com

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

Bu, yeni doğmuş sıçan yavruların üstün servikal ganglion (SCG) birincil sempatik nöronlar izole ve kültür nasıl açıklayan bir protokoldür.

Rat Üstün Servikal Ganglia (SCG) kültür Sempatik Nöronlar Protokolü

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

protocol-for-culturing-sympathetic-neurons-from-rat-superior-cervical

Research

JoVE Journal

Biology

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

22.6K Görüntüleme.  Columbia University. Bu, yeni doğmuş sıçan yavruların üstün servikal ganglion (SCG) birincil sempatik nöronlar izole ve kültür nasıl açıklayan bir protokoldür.

Video: Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

Mikroakışkan Aygıt compartmentalization E18 Kortikal Rat Nöronlar hazırlanması

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

Biyoloji

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

Nötrofil İzolasyon Protokolü

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

Kompartman Kültürleri Dorsal Kök Ganglia Nöronlar hazırlanması ve Bakım

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

Solucan Lab An Introduction to: Kültürleme Worms Mutagenez

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

Vahşi Tırtıllar gelen Hemocytes ayıklamak için basit bir protokol

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion.
Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.

Kanal Hücreleri Toplama kültür İlköğretim Rat İç Medüller

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Rat Yanal Kuyruk Ven gelen Kan Örnekleme

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

CD146 İzolasyonu<sup&gt; +</supSıçan Akcigerlerinden&gt; Resident Akciğer Mezenkimal Stromal hücreler

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Farelerden Pankreas Adacık İzolasyon için Basit Bir Yüksek Verimlilik Protokolü

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle biopsies are often collected for these endeavors. However, relatively few technical descriptions of biopsy procedures, outside of the commonly used musculus vastus lateralis, are available. Although the biopsy techniques are often adjusted to accommodate the characteristics of each muscle under study, few technical reports share these changes to the greater community. Thus, muscle tissue from human participants is often wasted as the operator reinvents the wheel. Expanding the available material on biopsies from a variety of muscles can reduce the incident of failed biopsies. This technical report describes a variation of the modified Bergstr&#246;m technique on the musculus tibialis anterior that limits fiber damage and provides fiber lengths adequate for mechanical evaluation. The surgery is an outpatient procedure that can be completed in an hour. The recovery period for this procedure is immediate for light activity (i.e., walking), up to three days for the resumption of normal physical activity, and about one week for wound care. The extracted tissue can be used for mechanical force experiments and here we present representative activation data. This protocol is appropriate for most collection purposes, potentially adaptable to other skeletal muscles, and may be improved by modifications to the collection needle.

This technical report describes a variation of the modified Bergstr&#246;m technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

Mekanik Değerlendirme için İnsan Musculus Tibialis Anterior Üstün Bölmesi İskelet Kas Biyopsileri Toplama

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle ...