Biz bu protokollerin gelişmesine katkıda Gelfand laboratuar üyelerine teşekkür ederim. Bu yayında bildirilen araştırma ödülü numarası BFI-2011-295 Udc için altında VIG ödül sayısı R01GM052111 altında Ulusal Sağlık Enstitüleri Genel Tıp Bilimleri Ulusal Enstitüsü tarafından ve Bask Devlet Eğitimi Bölümü, Üniversiteler ve Araştırma tarafından desteklenmiştir.

1.. Drosophila S2 hücrelerinin <ol><li> ConA kaplı lamelleri hazırlanması <ol><li> Seramik bir rafa lamelleri yerleştirin ve 1 saat kromik asit daldırma ile yıkayın. [DİKKAT: kromik asit aşındırıcı ve gözlerde tahriş, burun, boğaz ve cilt neden olabilir]. </li><li> Asit tamamen yıkanıp kadar sürekli 30 dakika dH 2 O ile tamamen lamelleri durulayın. </li><li> Let lamelleri hava kuru ve Con 30 dakika (dH 2 O 0.5 mg / ml çözelti) ile kat onları. </li><li> 15 dakika dH 2 O ile lamelleri durulayın ve onlara hava kuruması. ConA&#39;nın kaplı lamelleri 1 aya kadar saklanabilir. </li></ol></li><li> Hücreleri Kaplama <ol><li> S2 hücreleri katlanarak deney için gerekli hücre sayısına bağlı olarak, T25 veya T75 cm 2 şişelerinde büyümeye izin verin. </li><li> Hemasitometre kullanılarak hücre yoğunluğu sayılır. 1 ekleyin ml büyüme myumuşak bir 35 mm doku kültürü ConA kaplı lamel ile çanak ve içine edium (örneğin, böcek-Xpress) çanak ~ 1 x 10 5 hücre aktarın. Farklı hücrelerden süreçler örtüşmeyen çünkü bu hücre yoğunluğu görüntüleme için en uygunudur. </li><li> Hemen (2.5 uM CytoD nihai konsantrasyona) çanak 5 uM CytoD ile 1 ml orta ekleyin kaplama sonrası, işlemlerin oluşumunu uyarmak için. Görüntüleme önce 25 ° C&#39;de en az 2 saat boyunca hücrelerin inkübe edilmesi ile işlemlerin tam olarak gelişmesini sağlar. </li></ol></li></ol> 2.. Drosophila İlköğretim Nöron Kültürü <ol><li> Nöron kültürü için hazırlık <ol><li> (:, 5 ug / ml insülin, 100 ug / ml penisilin, 100 ug / ml streptomisin, 10 ug / ml tetrasiklin,% 20 fetal sığır serumu, 30 dakika boyunca 55 ° C &#39;de durdurulmuş ısı Schneider ortamı ile takviye edilmiş) Drosophila nöronal büyüme ortamı hazırlayın . Bu destekuygulandığın Schneider ortamı 6 ay boyunca 4 ° C &#39;de muhafaza edilebilir. </li><li> Drosophila embriyolar (22.5 g ağar, 12.0 g sükroz ve 750 ml H, 2 litrelik bir şişe içinde 2 O toplanması için elma suyu agar şişeler hazırlanması; 20-25 dakika için otoklav, 250 ml elma suyu ekleyin ve her Drosophila içine ~ 10 ml dökün flakon;) katılaşma sonrasında, pamuk topları ile şişeleri fiş ve gıda kurutma üzerinden önlemek için plastik gıda şal ile şişeleri sarın. Agar şişelere elma suyu 4 hafta içinde çok iyi sızdırmaz plastik torbalarda 4 ° C &#39;de muhafaza edilebilir. </li><li> ConA ile Coat asitle yıkanmış 25 mm daire lamelleri (Drosophila S2 hücreleri Protokolde detaylarını görmek) ve 10-15 dakika boyunca UV ışık altında sterilize. Bu lamelleri bir ay boyunca saklanabilir. </li></ol></li><li> Drosophila embriyonik nöron kültürü <ol><li> Keep genç erişkin embriyolar f toplama önce 1-2 gün boyunca kuru maya ile takviye elma suyu agar şişelere uçarNöron hazırlanması. </li><li> Toplama gününde, toplanan embriyoların senkronize etmek amacıyla, transfer yetişkin ışığında 1 saat precollection için taze elma suyu agar flakon uçar ve bu embriyolar atın. </li><li> Aktarım, 1 saat boyunca toplamak embriyolar ve embriyo verimini artırmak için karanlık hem de bir saat daha başka bir şişeye aktarın sinekler, yeni bir elma suyu agar şişesine uçar. </li><li> Elma suyu agar şişesinden yetişkin sinekler çıkarın ve embriyolar oda sıcaklığında (4-6 saat yaşındaki embriyolar evre 9-11 arasında) Evrelere başka bir 4 saat 9-11 geliştirmeye izin. </li><li> Viyallere fışkırmayı ve (en az 10 embriyolar iyi bir nöron hazırlık için gerekli olan) küçük bir fırça ile elma suyu agar embriyolar gevşeterek embriyolar çıkarın. </li><li> Suyu tahliye etmek için bir transfer pipet ile 100 mikron naylon örgü hücre süzgeç embriyolar transfer (Drosophila embriyo yıkama tamponu,% 0.4 NaCl,% 0.03 Triton transfer pipet Ön yıkamaX-100). </li><li> Küçük bir fırça ile süzgeçten embriyoların toplayın ve 1 ml Drosophila embriyo yıkaması (Not: fırça veya pipet uçları kullanılarak bir agar ya da pamuk lifleri temiz) ile dolu bir 1.5 ml mikrotüp içine aktarın. Bu protokolde, 1.5 ml Mikrotüp eşleşen tek pelet havan tokmağı ile birlikte gelir. </li><li> Drosophila embriyo yıkama 3x embriyolar yıkayın. </li><li> 10 dakika boyunca taze 01:01 ticari çamaşır suyu çözeltisi ve% 95 etanol içinde embriyolar Dechorionate [DİKKAT: ağartma tahriş Gözleri, burun, boğaz ve deriyi neden olabilir]. </li><li> Doku kültürü kaputu taşı ve desteklenmiş Schneider orta embriyolar 2x yıkayın. </li><li> Embriyolar ile tüp 200-300 ul orta bırakın ve yavaşça mekanik (10-15x eziyet)% 70 etanol ile sterilize edilmiştir Kimble Chase tek pelet pestles kullanarak dechorionated embriyolar ayrışır. </li><li> 2 dakika ve 20 tra xg&#39;de homojenleştirilmiş karışım santrifüj, yeni bir tüpe yüzer nsfer. [Bu adım isteğe bağlıdır, bu embriyo enkaz kaldırmak için yardımcı olur] </li><li> Hücreler pellet haline getirmek için 2 dakika boyunca 550 x g&#39;de santrifüje süpernatan. </li><li> 2 dakika boyunca 550 x g hızında tekrar Schneider orta ve spin hücreleri aşağı desteklenmiş 500 ul pelletini, bir kez resüspansiyon-iplik döngüsü tekrarlayın. </li><li> Son olarak Schneider ortamı ilave ~ 100 ul pelet tekrar süspansiyon ve steril bir 35 mm Petri kabı, bir 25 mm ConA kaplı lamel üzerine hücreleri plaka. Hücre bağlanmasına olanak sağlamak için 10 dakika boyunca inkübe edin. </li><li> Bağlı hücreleri ile lamel daldırın 5 uM CytoD ile takviye Schneider ortamı 2 ml ekleyin. </li><li> Görüntüleme gecede önce nemlendirilmiş bir 25 º C inkübatör inkübe çanak. </li></ol></li></ol>

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Yazarlar, hiçbir rakip mali çıkarlarını olmadığını beyan ederim.

Burada Drosophila S2 hücreleri ve primer nöron kültürü mikrotübül bağımlı kargo taşımacılığı eğitimi için ayrıntılı protokoller mevcut. Hem S2 süreçler ve nevrotiler organel ulaşım için parça olarak hizmet bohça mikrotübülüsleri diziler tarafından doldurulur yapılardır. Iki strateji genellikle etiket organellere kullanılır: kültürlere doğrudan ilave floresan boyalar ile bir organel-hedefleyen dizisi ya da boyama ile bir floresan proteini kodlay...

Drosophila S2 hücreler birçok hücresel süreçleri incelemek için artan popülerlik kazanmıştır. Bu hücreler başlangıçta OregonR Drosophila melanogaster tripsinlendi geç dönem embriyo elde edildi ve 1.. Drosophila S2 hücreleri diğer hücre hatları üzerinden pek çok avantajı sunmaktadır makrofaj gibi özelliklerini korumak edildi. Bu CO2 olmadan 25 ° C&#39;de kültürlenir ve ısıtma veya gaz değişimi ihtiyacı olmadan, oda sıcaklığında saatlerce görüntülenebilir. Daha da önemlisi, Drosophila S2 hücreleri, ilgi konusu olan proteinlerin yüksek verimlilik sağlayan demonte RNAi tedaviye çok duyarlıdır. S2 hücreleri yüksek oranda transfectable ve kararlı hücre soyları ilgi konusu olan proteinlerin kararlı ektopik ifade izin vermek için en az dört hafta içinde seçilebilir. S2 hücreleri normalde ya büyümeye gevşek bağlı olduğu, ancak, bir şişe ya da süspansiyon içinde yayılmaz. Bir bitki lektin Conca ile önceden kaplanmış lameller üzerine yaymak için indüklenebilirnavalin A (ConA). Yayılmış hücrelerin çevresi özellikle incedir ve bu nedenle canlı hücre mikroskopi için idealdir. S2 hücreleri kültür, RNAi tedavisi, canlı ışık görüntüleme ve immün için ayrıntılı protokoller literatür 2,3 bulunabilir. Bizim laboratuvar. Gibi sitokalasin D gibi F-aktin depolimerize veya sunucularının herhangi bir ilaç, (CytoD) ile muamele Drosophila S2 hücrelerinin homojen bir polarize mikrotübül 4 ile doldurulmuş uzun süreçler büyümesi mikrotübül göre yük taşıma çalışmak için bir model sistem olarak S2 hücreleri benimsemiştir -10, bu mikrotübül diziler boyunca kargo hareketini incelemek için ideal bir sistem yapar. Bu süreçlerin farklı ulaşım parametreleri (. Yani yörüngeleri uzunlukları, hızlar, yön vb) kolayca otomatik partikül izleme yazılımı, DiaTrack (Semasopht kullanılarak ölçülebilir: http://www.microscopy.info/Organization/Details/4423; Dr . Pascal Vallotton: http://www.csiro.au/en/Organisation-Structure/Divisions/Mathematics-Informatics-and-Statistics/PascalVallotton.aspx # a4). Bu özellikler S2 hücreleri doku kültürü hücrelerin mikrotübüllerinde birlikte kargo taşımacılığı eğitimi için cazip bir sistem yapmak. S2 hücrelerinde Pilot Deneyler Drosophila birincil nöronlar kargo taşımacılığı çalışmalar fizyolojik olarak önemli bir örnek için uzatılabilir. S2 hücrelerine benzer şekilde, ayrışmış embriyolardan kültür nöronları gibi boyalar ve inhibitörleri gibi küçük moleküllere karşı geçirgen olan ve organel taşıma yüksek çözünürlüklü canlı görüntüleme oda sıcaklığında nöronlarda gerçekleştirilebilir. Farklı genetik geçmişleri ile Drosophila kullanarak, nakavtla (genetik kayıp fonksiyon-mutasyonlar) primer nöronlar gen fonksiyonunu inceleyebilirsiniz, demonte (dsRNA enjeksiyon ya da ifade) veya ektopik ifadesi (transgenik sinekler). Özellikle, CytoD tedavi kullanarak aktin filamentler parçalanması nörit gelişimini engellemez, onun yerine daha hızlı büyür ve böylece biz mikrotübül-depende eğitim görebilirsinizaktin etkisi olmadan CytoD ile muamele edilmiş nöronlarda nt organel nakil 11, parçalanmış olması gerekir. Bu nedenle, birincil nöronal kültürlerin bir fizyolojik ilgili sisteme 10,12 kargo taşımacılığı çalışma için harika bir sistem kılan, doku kültürü hücreleri avantajlarını birleştirmek ve genetik sinek. Birkaç ailesel nörodejeneratif hastalıkların yol açtığı veya kargo taşımacılığı kusurları (örneğin Parkinson hastalığı 13 ile ilişkili olabilir bu yana, Huntington hastalığı 14, Alzheimer Hastalığı 15,16, Amyotrofik Lateral Skleroz / ALS 17, HMN7B ve Perry sendromu 18,19 ve Spinoserebellar ataksi tip 5/SCA5 20), ve primer nöron kültürlerinden mikrotübüle bağlı taşıtlarda, bu hastalıklara neden olan belirli bir mutasyon etkilerinin analizinde yararlı olabilir. Son olarak, mikrotübül-bağlı taşıma önemli özellikleri de memeli hayvanlarda ve sinekler arasında muhafaza, fakat <em&gt; Drosophila genetik ve hücre kültürü, normal ve patolojik koşullarda organel taşıma temel yönlerini incelemek için kültürlenmiş Drosophila hücrelerinin kullanımını haklı, daha az pahalı ve zaman alıcıdır.

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organelle transport in cultured drosophila cells: s2 cell line and primary neurons.

Drosophila S2 hücrelerinin homojen bir polarize mikrotübüller ile dolu bir aktin-depolimerizasyon ilaç formu uzun dallanmamış süreçlerin mevcudiyetinde bir lamel üzerine kaplanmıştır. Organelleri mikrotübül motorları ile bu işlemler boyunca hareket eder. Kolay bakım, RNAi protein knock-down ve istikrarlı hücre hatları oluşturmak için verimli bir prosedüre yüksek hassasiyet Drosophila S2 hücreleri canlı görüntüleme ile kargo taşımacılığı çalışma için ideal bir model sistemi yapmak. Ayrışmış Drosophila embriyolardan kültür primer nöronlarda aksonal taşıma araçları: S2 hücreleri ile elde edilen sonuçlar ayrıca, daha fazla fizyolojik olarak uygun bir sistemin uygulanabilir. Kültürlü nöronlar S2 işlemlere benzer birlikte mikrotübüllerin, dolu uzun nöritler büyür. Gibi S2 hücrelerindeki, kültüre edilmiş nöronlarda organeller ya organel-spesifik floresan boyalar veya DNA tarafından kodlanan bir organel fluoresan işaretleyiciler kullanılarak görüntülenebilir erken embriyolar içine enjekte edilen ya da transgen ekspreseic uçar. Bu nedenle, organel ulaşım kolaylıkla yaşayan görüntüleme kullanarak cam lamelleri kültüre nöronlar kaydedilebilir. Burada Drosophila S2 hücreleri ve primer nöronlar kargo taşımacılığı kültüre ve görselleştirmek için prosedürler açıklanmaktadır. Biz bu protokoller kargo taşımacılığı eğitim laboratuarları için her iki sistemin erişilebilir hale inanıyoruz.

Düz yuvarlak &quot;pancake&quot; şekli (Şekil 1A ve 1C) içine ConA&#39;nın kaplı lamel formada bağlı Drosophila S2 hücreleri. S2 hücreleri CytoD mevcudiyetinde lamelleri kaplama ve 2-3 saat boyunca yayılmasına izin verilir, ancak, bunlar paralel mikrotübül (Şekil 1 B ve 1 D) ile doldurulmuş ince uzun bir süreç oluştururlar. 7 out artı-biter bu mikrotübülüsleri üniforma kutba sahip. Drosophila S2 stabi...

Watch this Scientific Journal Video about Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons. at JoVE.com

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 hücreleri ve kültürlenmiş nöronların in vivo olarak motor tahrikli bir organel taşıma görüntüleme için büyük sistemlerdir. Burada, her iki hücre kültür tipleri için, görüntüleme ve taşıma analiz protokolleri ayrıntılı tarif etmektedir.

Kültürlü organel Ulaşım<em&gt; Drosophila</em&gt; Hücreler: S2 Hücre Hattı ve İlköğretim Nöronlar.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

organelle-transport-cultured-drosophila-cells-s2-cell-line-primary

Research

JoVE Journal

Biology

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

13.6K Görüntüleme.  Feinberg School of Medicine, Northwestern University.  Drosophila S2 hücreleri ve kültürlenmiş nöronların in vivo olarak motor tahrikli bir organel taşıma görüntüleme için büyük sistemlerdir. Burada, her iki hücre kültür tipleri için, görüntüleme ve taşıma analiz protokolleri ayrıntılı tarif etmektedir. 

Video: Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

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 ...

Biyoloji

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

İlköğretim Kültür Hipokampal Nöronlar Yoğun çekirdekli veziküller Canlı Görüntüleme

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Preparation of Drosophila S2 cells for Light Microscopy

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos1, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells.
S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media2. In addition, they grow optimally and quickly at 21-24&deg;C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks.
Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)3. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher's choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.

Preparation of Drosophila S2 cells for Light Microscopy

Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.

Hazırlanması Drosophila Işık Mikroskopi S2 hücreleri

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

Kültür İlköğretim Fare Embriyonik Fibroblastlar Sinyal Molekülleri Adenovirüs aracılı Genetik Kaldırma

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

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 ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Sıçan Karaciğer Hücreleri İzolasyon ve İlköğretim Kültür

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Görüntüleme Yerel Ca<sup&gt; 2+</sup&gt; Kültürlenmiş memeli hücrelerinde sinyaller

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Generating a &#34;Humanized&#34; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper distribution of the genetic material requires that sister kinetochores on every chromosome become bioriented by attaching to microtubules from opposite spindle poles before progressing into anaphase. However, erroneous, non-bioriented attachment states are common and cellular pathways exist to both detect and correct such attachments during cell division. The process by which improper kinetochore-microtubule interactions are destabilized is referred to as error correction. To study error correction in living cells, incorrect attachments are purposely generated via chemical inhibition of kinesin-5 motor, which leads to monopolar spindle assembly, and the transition from mal-orientation to biorientation is observed following drug washout. The large number of chromosomes in many model tissue culture cell types poses a challenge in observing individual error correction events. Drosophila S2 cells are better subjects for such studies as they possess as few as 4 pairs of chromosomes. However, small molecule kinesin-5 inhibitors are ineffective against Drosophila kinesin-5 (Klp61F). Here we describe how to build a Drosophila cell line that effectively replaces Klp61F with human kinesin-5, which renders the cells sensitive to pharmacological inhibition of the motor and suitable for use in the cell-based error correction assay.

Generating a &quot;Humanized&quot; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

This protocol describes how to generate a Drosophila S2 cell line that is sensitive to small molecule inhibitors of kinesin-5. The use of these cells in a cell-based error correction assay is also outlined.

Bir &quot;humanize&quot; oluşturuluyor<em&gt; Drosophila</emKinesin-5 Farmakolojik inhibisyonu için&gt; S2 Hücre Hattı Duyarlı

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Glikoz Alımı Ölçümü ve İnsülin Uyarıcısına Yanıt<em&gt; In vitro</em&gt; Kültürlü İnsan Primer Miyotları

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory cells, EC cells sense luminal stimuli and convert them into serotonin (5-hyroxytryptamine, 5-HT) release events. However, the electrophysiology of these cells is poorly understood because they are difficult to culture and to identify. The method presented in this paper outlines primary EC cell cultures optimized for single cell electrophysiology. This protocol utilizes a transgenic cyan fluorescent protein (CFP) reporter to identify mouse EC cells in mixed primary cultures, advancing the approach to obtaining high-quality recordings of whole cell electrophysiology in voltage- and current-clamp modes.

Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology.

Tüm cep telefonu Elektrofizyoloji birincil kültürlü fare Enterochromaffin hücre

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory ...