Bu çalışma, Ulusal Sağlık Enstitüsü, hibe NS27501 tarafından desteklenen DKOD. Bu iş için ek destek, DKOD HHMI Profesör Programı aracılığıyla destek UC Irvine hibe sağlanmıştır.

Kültür gün önce hazırlıklar: <ol><li> Steril diseksiyon çözüm olun. </li><li> Steril DMEM olun ve 10 ml alikotları tutmak, 4 ° C 2 hafta boyunca. </li><li> 1 ay için 50 veya 100 ul alikotları steril DDM2 takviyeleri ve donma olun. </li><li> CONA / laminin olun. </li><li> Kat lamelleri. İsteğe Bağlı: 4 ay kadar donmuş steril Cnbm ve mağaza olun. </li></ol> Kültür, günü I. laminer akış kaput Enzim Çözümü (ES) hazırlayın <ol><li> 5 ml, 15 ml santrifüj tüpüne çözüm diseksiyon (DS) koyun. </li><li> 0.6 ml Eppendorf 0.8 mg L-Sistein tartılır ve DS 150 ul ekleyin. Kristaller gidene dek, yukarı ve aşağı Pipet. </li><li> 150 ml 5 ml DS DS / Sistein ekleyin ve iyice karıştırın. </li><li> 50 U Papain (adet) ekleyin. </li><li> 7 ml 0.1 N Na OH ekleyin ve iyice karıştırın. Çözüm 30 dakika içinde açık olacak. aktive edildiğinde. </li><li> 0,2 mm steril 1,5 ml vidalı kapaklı mikrofuge&#39;de tüp içine şırınga filtresi ile filtre enzim çözeltisi Papain: 5 ml DS 50 U Wanna. Her parti, biraz farklı, bu yüzden ne kadar hesaplamak gerekir: 37.3 mg / ml ve 28.3 U / mg 37.3 x 28.3 = 1055,59 U / ml (1000 ml) 50 U = 47.4 ml </li></ol> II. DDM2 medya yapın Kültür, günü: DDM2 yapmak için aşağıdaki takviyeleri eklemek Için 10 ml DMEM, sadece kültür önce takviyeleri ekleyin: <ol><li> 100μl Transferrin </li><li> 100μl Putrescine </li><li> 100μl Selenyum </li><li> 100μl Progesteron </li><li> 50μl İnsülin </li><li> 10μl 20-Hydroxyecdysone </li></ol> Not: planlanan tüm kültürlerde (her beyin ayrı bir 35 mm Petri kabında tek bir lamel, medya 1.5 ms / kültür kaplama) için yeterli DDM2 olun Adım III-VI yapılan steril olmayan bir laboratuvar tezgahı ve 45 dakika veya daha kısa sürede tamamlanması gerekmektedir. III. Pupa Koleksiyonu <ol><li> Diseksiyon mikroskop altında 10-15 flakon taraftan pupa seçin. </li><li> Pupa, kuru bir 35 mm Petri kabında kapak yerleştirin. </li></ol> Not: Biz genellikle puparium formasyonu (APF) sonra 55-78 saat arasında pupa beyinlerini kullanmak. Neurite akıbet genel düzeyi en eski pupa biraz daha düşük iken baş kapsül, yıkılmaya yüz çünkü genç pupa beyin incelemek için biraz zordur. Canton-S wild tip gerginlik, bu aşamalar, hafif hafif kırmızımsı kahverengi pigmentli gözlerinden başka bir yerde çok az pigment ile tanınır. IV. Diseksiyon mikroskobu altında Decapitation <ol><li> Yeri sonraki iki damla steril diseksiyon çözüm Petri kabı kapağı, pupa. </li><li> Yavaşça sol elinde ince forseps ile tek bir pupa tutun ve ön pupa sonunda manikür kaldırmak için sağ elinde bir 1 cc şırınga, 27 gauge bir şırınga iğne kullanın. </li><li> Forseps ile pupa hafifçe sıkın ve baş çıktığında boyun koparmak için 27 gauge iğne kullanınız. </li><li> Iğne ucu veya forseps ile çözüm diseksiyon bırak baş aktarın. </li><li> 10-15 baş tek bir damla kadar tekrarlayın. </li></ol> Diseksiyon mikroskobu altında kafa beyin V. Kaldırma <ol><li> Her el, 27 gauge iğne ile 1 cc şırınga tutun. </li><li> Burnumun siz ve başın dorsal yüzeyi kuzeye bakacak şekilde çözüm diseksiyon açılan sinek kafa pozisyonu kullanın. </li><li> Baş aşağı pin ve ventral dorsal yüzeyine gider sağ gözünde bir yarık yapma hakkını saklı iğne kullanmak için burnumun içine sol iğne yerleştirin. </li><li> Burnumun bölgede sol yanında doğru bir iğne takın ve sonra beynin sağ gözü yarık doğru iterek manikür, sol gözün üzerine hafifçe bastırmak için sol iğne kullanınız. Genellikle optik lo hem beyin, nispeten bozulmamış, sağda yarık ortaya gerektiğinibes de hala bağlı ve bazen pigmentli göz dokusu. </li><li> Beyin, yeni bir steril, 35 mm Petri kabı, steril diseksiyon serum fizyolojik damla iğne ve transfer sırtına doğru itin. </li><li> Her genotip için 10-15 beyinleri toplayın. </li></ol> VI. Kaldırılması, optik loblar ve enzim tedavisi <ol><li> 27 gauge iğne ile 1 cc şırınga tutun ve her elin tüm beyinleri diseksiyon çözüm açılan küçük bir alanda toplamak için bu </li><li> Böylece her bir beyin doku kültürü kalan merkezi beyin bölgesi optik loblar kaldırmak için kesici aletler şırınga kullanın. </li><li> 5 ul 20 ul Pipetman, Eppendorf tüpüne 1 ml steril papain çözüm içeren tek bir genotip tüm beyinleri aktarmak için kullanın. </li><li> Brain için papain 60 devirde bir rotator seti 10-15 dakika inkübe edildi. </li><li> 3000 rpm&#39;de 3 dakika boyunca santrifüjleyin. </li></ol> Steril laminer akış kaputu İçinde tüm adımları doku havaya maruz kalan adımları için laminer akış kaput yapılmalıdır. VII. Yıkama <ol><li> Enzim çözeltisi çıkarın ve steril diseksiyon çözümü ile değiştirin. </li><li> 3000 rpm&#39;de 3 dakika boyunca santrifüjleyin. </li><li> Steril diseksiyon çözümü ile bir toplam 3 kez yıkayın. </li><li> Diseksiyon salin çıkarın ve DDM2 ile değiştirin. </li><li> Santrifüj ve DDM2 ile toplam 2 kez yıkayın. </li><li> Steril bir 35 mm Petri kabında Transfer beyin ve medya. </li></ol> VIII. Diseksiyon mikroskobu altında ezerek toz haline getirme ve Kaplama <ol><li> DDM2 bir lamel merkezinde 5 ul yerleştirin. </li><li> Sarı bir ucu ile DDM2 Bu düşüşün 1 beyin aktarın. </li><li> Mikroskop altında beyin zar kadar küçük parçalar halinde (&lt;1 dakikada almalıdır), 27 gauge iğne kullanın. </li><li> Görsel rehberliği altında, büyük parçalar pipetlemeyin ucu içine yavaşça emilir ve ortama geri sınırdışı böylece iyi bir noktaya çekti damlalıklı cam ucunu kırmak. Biz 100 ul hematokrit kapiller cam ile standart olarak gelir ağız emme boruları kullanın. Not: medya kabarcıkları almak için özen ve kullanmak için en iyi boyutu pipetlemeyin ampirik olarak belirlemek gerekir. İyi olanlar, yaklaşık 30 saniye / beyin, bazı bireysel hücrelerin ve hala bazı büyük öbekler beyin ayırmak izin verir. Yüksek güçte bu baktığınızda, nöronların yaklaşık% 10-20 hala kalan bazı işlemler var ve hala bazı büyük öbekler olacak. Çok ayrıştırmaları varsa, o zaman tüm hücreleri yuvarlak olacak ve onlar hayatta olmaz bu kadar zarar görmüş olacak. Bu adım, uygulama alır ve hızlı bir şekilde değiştirebilirsiniz büyük bir yüzey-hacim oranı ve DDM2 açılan osmolalitesi ve pH olmadığından hızlı bir şekilde yapılmalıdır. Doku, mümkün olduğunca çabuk CO 2 inkübatör yerleştirilmesi gerekiyor . </li><li> &quot;Genotip, tarih ve saat&quot;: petri kapak yaz. </li><li> , 100 mm petri, 35 mm petri (ıslak kimwipe) koyun ve 30 dakika CO 2 inkübatör yerleşmek izin . </li><li> DDM2 1.5 ml 35 mm tabak Sel. </li><li> % 5 CO 2 inkübatör (23 ° C) inkübatör kültürleri tutun. Not: Eğer bir soğutma CO 2 inkübatör erişim yoksa, standart bir memeli doku kültürü inkübatör kullanımı ve serin bir odada ve ısı 23 ya koymak ° C veya laboratuarda koymak, geçici kapatmak ve kullanmak kuluçka altındaki ortam etrafında düzenleyen geçici bir tepsi buz. </li></ol> IX. Besleme Hücreler <ol><li> CNBM/B27: 1 Gün (ertesi gün), 03:01 DDM2 yapmak CNBM/B27 0.5 ml (Şartlandırılmış Medya) eklemek. </li><li> CNBM/B27: Günlük 5 03:01 DDM2 için 1 ml medya değiştirin. </li><li> CNBM/B27 her 4-5 gün: 03:01 DDM2 besleyen hücrelerin tutun. Not: Kültürleri nöronal olmayan klimalı medya olmadan yetiştirilir ama nöronlar biraz daha kırılgan görünür ve elektrofizyoloji deneylerde kullanılmak için daha zor olabilir. </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. , (1991).</li><li>Rohrbough, J., O'Dowd, D. K., Baines, R. A., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+bases+of+behavioral+plasticity:+Establishing+and+Modifying+synaptic+circuits+in+the+Drosophila+Genetic+System.">Cellular bases of behavioral plasticity: Establishing and Modifying synaptic circuits in the Drosophila Genetic System.</a> J. Neurobiol. 54, 254-271 (2003).</li><li>O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-gated+currents+and+firing+properties+of+embryonic+Drosophila+neurons+grown+in+a+chemically+defined+medium.">Voltage-gated currents and firing properties of embryonic Drosophila neurons grown in a chemically defined medium.</a> J. Neurobiol. 27, 113-126 (1995).</li><li>Lee, D., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+excitatory+synaptic+transmission+mediated+by+nAChR+in+cultured+Drosophila+neurons.">Fast excitatory synaptic transmission mediated by nAChR in cultured Drosophila neurons.</a> J. Neurosci. 19, 5311-5321 (1999).</li><li>Su, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+synaptic+currents+in+Drosophila+mushroom+body+Kenyon+cells+are+mediated+by+alpha-bungarotoxin-sensitive+nAChRs+and+picrotoxin-sensitive+GABA+receptors.">Fast synaptic currents in Drosophila mushroom body Kenyon cells are mediated by alpha-bungarotoxin-sensitive nAChRs and picrotoxin-sensitive GABA receptors.</a> J. Neurosci. 23, 9246-9253 (2003).</li><li>Jiang, S. A., Campusano, J. M., Su, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+mushroom+body+Kenyon+cells+generate+spontaneous+calcium+transients+mediated+by+PLTX-sensitive+calcium+channels.">Drosophila mushroom body Kenyon cells generate spontaneous calcium transients mediated by PLTX-sensitive calcium channels.</a> J. Neurophysiol. 94, 491-500 (2005).</li><li>Campusano, J. M., Su, H., Jiang, S. A., Sicaeros, B., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nAChR-mediated+calcium+responses+and+plasticity+in+Drosophila+Kenyon+cells.">nAChR-mediated calcium responses and plasticity in Drosophila Kenyon cells.</a> Develop Neurobiol. 67, 1520-1532 (2007).</li><li>Oh, H. -. W., Campusano, J. M., Hilgenberg, L. G. W., Sun, X., Smith, M. A., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+chemical+synapses+and+gap+junctions+between+Drosophila+brain+neurons+in+culture.">Ultrastructural analysis of chemical synapses and gap junctions between Drosophila brain neurons in culture.</a> Develop Neurobiol. , (2007).</li></ol>

Neurites uzatmak ve fonksiyonel sinaptik bağlantıları oluşturmak embriyonik / postnatal kemirgenler beyinleri hasat nöronlar primer hücre kültürü yetiştirilebilir. Bu kültürlerin hazırlanması için Yöntemleri iyi kurulmuş ve kemirgen nöronal kültürlerin çalışmalar, sinaps oluşumu ve işlevi düzenlenmesi (Banker ve Goslin, 1991) yer alan genler ve çevresel faktörlerin belirlenmesi kritik bir rol oynamıştır. Türlerin çeşitli böcek nöron kültüründe yetişen olabilir, fonksiyonel kültür sinaptik bağlantı...

Kaplama lameller: 60 mm petri otoklavlanmış lamelleri koyun. Con pipetleyin 5 ul her lamel merkezi üzerine A / Laminin karışımı. 2 saat boyunca 37 ° C inkübatör yerleştirin. 3x lamelleri ul otoklava su, her biri 100 ile durulayın. Steril bir yeniden süspanse bağlı vakum kullanın. Son durulama sırasında forseps ile lamel pick up ve her iki tarafın da kuru. 35mm Petri kabı lamel aktarın. Bir aya kadar oda sıcaklığında saklayın.

primary neuronal cultures from the brains of late stage drosophila pupae

Bu video, geç dönem Drosophila pupa beyinleri primer nöronal kültürlerin hazırlanmasında gösteriyor. Prosedür puparium oluşumundan sonra 70-78 saat hayvanların beyinlerinin kaldırılması ile başlar. Izole beyinleri serum büyüme ortamında birkaç yıkar takip papain kısa inkübasyondan sonra gösterilmiştir. Bir lamel medya 5 ul açılan her bir beyin mekanik ayrılma süreci gösterilmektedir. Post-mitotik nöronların akson ve dendritler disosiasyon sırasında soma yakın sheered ama nöronlar kaplama bir kaç saat içinde süreçleri yeniden başlar. Görüntüler 2 gün canlı kültürler göstermektedir. Nöronlar kültüründe ilk haftasında süreçlerini ayrıntılı olarak devam etmektedir. Spesifik nöronal nüfus, kültür, GFP veya RFP floresan belirteçleri doku belirli bir ifade sürücü GAL4 hatları kullanarak tespit edilebilir. Tüm hücre kayıtları kültürlü nöronların fonksiyonel, kendiliğinden aktif ve GABAerjik kolinerjik sinaps oluşturur göstermiştir. Kısa bir video segment, kültürlü nöronların bir tabak içinde kendiliğinden kalsiyum transientler ve nikotin uyarılmış kalsiyum tepkileri izlemek için bir kalsiyum gösterge boyası olarak Fura-2 kullanarak kültürlü nöronlarda kalsiyum dinamiklerini göstermektedir. Bunlar pupa beyin kültürleri, genetik ve farmakolojik araçları merkezi sinaps oluşumu ve fonksiyonunu etkileyen iç ve dış faktörleri tanımlamak için kullanılabilecek yararlı bir model sistem.

Watch this Scientific Journal Video about Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae at JoVE.com

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

Bu video, geç dönem Drosophila pupa beyinleri primer nöronal kültürlerin hazırlanmasında gösteriyor. Canlı kültürlerin Görüntüleme Fura-2 kullanarak kalsiyum düzeyleri neurite akıbet ve görüntüleme göstermektedir.

Geç Stage Drosophila pupa Brain İlköğretim Nöronal Kültürler

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

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Research

JoVE Journal

Biology

15.0K Görüntüleme.  University of California, Irvine (UCI). Bu video, geç dönem Drosophila pupa beyinleri primer nöronal kültürlerin hazırlanmasında gösteriyor. Canlı kültürlerin Görüntüleme Fura-2 kullanarak kalsiyum düzeyleri neurite akıbet ve görüntüleme göstermektedir.

Video: Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

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

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

Nöronal Kültürler Midgastrula Sahne Drosophila Embriyolar hazırlanması

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

Biyoloji

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

Büyüme Konileri Yüksek Çözünürlüklü Görüntüleme için Aplysia Nöronal Hücre Kültürleri

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

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

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

Kemirgen Yenidoğanlar İlköğretim grubuyla orta beyin dopamin Hücre Kültürleri

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

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Hazırlık Elektroporasyon murin Kemik İliği itibaren İlköğretim Hematopoetik Hücre Kültürleri

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

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

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

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

Hazırlanması Aplysia Duyusal-motor Nöronal Hücre Kültürleri

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

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

Geliştirilmesi ve Yetişkin hazırlanması Drosophila Brain ve Canlı Görüntüleme için Retinae

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

Kemirgenler itibaren İlköğretim Yetişkin Fibroblast Kültürleri kurulması

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Birincil Hücre Kültürleri Drosophila Gastrula Embriyolar

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

İlköğretim İnsan fibroblast Kültürler gelen naif Erişkin Kök Hücre İzolasyon

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

Of Utility Sahne özgü Orta-geç<em&gt; Drosophila</em&gt; Folikül İzolasyon

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...