JB Damon Runyon Kanser Araştırma Vakfı Bursu DRG-1716-1702 tarafından desteklenmiştir. JZ NIH / NIGMS Bursu F32 GM082174-02 tarafından desteklenmiştir. NP Howard Hughes Tıp Enstitüsü&#39;nden bir araştırmacı.

1. Sinek kafesleri hazırlanması <ol><li> Drosophila (Oregon-R ya da herhangi bir genotip gibi yabani tip suşlar), 32-oz böcek bardak gibi uygun kaplarda büyütün. </li><li> Büyük embriyo toplama kafesleri Transfer yeni eclosed sinekler veya nüfus kafesleri uçmak. </li><li> ~ 25 bir sıcaklık ile bir odaya kafesleri Taşı ° C ve nem ~ 65% 12 saatlik ışık ve senkron embriyoların toplanmasını kolaylaştırmak için, 12 saat karanlık bir döngüsü. </li><li> Onlara pekmez plakalar üzerinde çizikler maya yapıştırın öldürdü beslenme kafeslerde sinekler koruyun. 2-3 günde bir kez pekmez levhalar (öldürüldü maya yapıştır ile) değiştirin d. </li></ol> 2. Senkron embriyo toplanması <ol><li> Birincil hücre hazırlık günü, 1-2 saat öncesi yatıyordu süre yapıştırın öldürüldü maya ile kaplı uçar wth taze plakaları beslenirler. </li><li> Sadece 12 saat karanlık döngüsü önce, az miktarda maya hamur çizgili taze pekmez plakaları ile ön yatıyordu plakaları değiştirin. Asenkron büyük embriyoları içeren öncesi yatıyordu toplanması, atın. </li><li> 1-2 saat bir süre için toplayın embriyolar </li><li> 5-6 saat boyunca 25, embriyo ve mağaza nispeten senkron nüfus içeren plakalar ° C çıkarın Embriyolar bu zamanda, ileri gastrula aşamada olacak. </li></ol> 3. Toplama ve embriyo yıkama <ol><li> Embriyoların altındaki en güzel örgü elek toplamak, böylece, ıslak bir fırça ile plakalar embriyoların gevşetin ve yığılmış elekler ılık musluk suyu ile iyice durulayın. Mümkün olduğu kadar çok maya ve sinek enkaz kaldırmak için bir kaç dakika durulayın. </li><li> İpucu embriyoların bir tarafında birikir elekler ve bir dechorionation sepet içine yavaşça elek onları yıkayın. </li><li> Embriyoların kurumasına izin vermeden doku kültürü kaputu alana taşıyın. </li></ol> 4. Embriyo hücrelerinin Homojenizasyon ve kaplama <ol><li> Sterilize ve embriyoların% 50 (hacim / hacim) 5-10 dakika çamaşır suyu çeker dechorionate. Sepet duvardan embriyoların% 70 (hacim / hacim) etanol ile durulayın. Alternatif olarak, etanol tedavi öncesi, distile su ile çamaşır suyu embriyolar kapalı yıkayın. Bu noktadan itibaren embriyolar steril şartlarda ve doku kültürü kaputu içinde ele alınmalıdır. </li><li> Otoklavlanmış distile su ile çamaşır suyu ve / veya etanol kaldırmak için embriyoların iyice durulayın. </li><li> Dechorionation adım sırasında, embriyoların miktarına göre oda sıcaklığında M3 orta uygun hacmi ile Dounce Homojenizatör doldurun. Embriyo ve medya hacmi arasındaki oranı 0.5 ml embriyolar üzerinde olmamalı: 40 ml medya (veya ~ 100-200 embriyolar / ml). </li><li> Batığın embriyoların M3 medya için yeterli miktarda steril bir beher durulanır embriyolar yerleştirin. Embriyolar 2-5 dakika bekletin edelim. </li><li> Dechorionation sepeti sökün ve Nitex örgü kuru steril kağıt havlu ile kurulayın. </li><li> Homojenizatör embriyolar steril bir fırça ile aktarın. </li><li> Tüpün alt ulaşmadan tüm embriyolar askıya alınır kadar gevşek bir havaneli kullanarak kısa vuruş ile hafifçe karıştırılır. Sonra kibarca ama kesin bir tavırla, tam sekiz vuruş ile homojenize. Aşağı yukarı doğru hareket eder ve havaneli bükmeyin. </li><li> 50 ml steril konik santrifüj tüpü içine dökme veya pipetleme ya Homojenat transfer ve tüp kap. </li><li> 40g, pelet oda sıcaklığında doku artıkları, büyük hücre kümeleri ve vitelline membranlar 10 dakika santrifüjleyin. Temiz bir tüp hücreleri ve yumurta sarısı içeren süpernatant aktarın ve 5 dakika süreyle santrifüj adımı tekrarlayın. </li><li> Hücreler dökülerek veya pelet, 360g oda sıcaklığında 10 dakika için temiz bir tüp ve spin pipetleme süpernatantı dikkatlice aktarın. Süpernatantı atın. 10 ml kültür ortamı hücre pelet (RNAi deneyler için serum orta) tekrar </li><li> Hücre yoğunluğunu tahmin etmek için bir hemasitometre kullanarak canlı hücreler güvenin. Bu arada, pelet hücreleri 4.10 adımı tekrarlayın. </li><li> Istenen konsantrasyon hücreleri yeniden süspanse edin. Başlatmak için, bir dizi 1 ~ 5 x10 6 hücre / ml ve 1,7 - 2,5 x10 5 hücre / cm 2 plaka deneyin . </li></ol> 5. 384 plaka yetiştirilen hücreler için İlköğretim-hücre RNAi <ol><li> ~ 250 ng 384 plaka de ortalama bir final konsantrasyonda her kuyuya dsRNAs 5 ul koyun. </li><li> Çok kanallı pipet kullanın, 10 ml (1 ~ 4x106 hücre / ml) 384-plaka her yanı farklı bir dsRNA içeren bir serum serbest M3 orta başına birincil hücreler dağıtmak. Alternatif olarak, hücre konfokal görüntüleme için 8-iyi cam oda slaytlar üzerinde yetiştirilebilir. </li><li> 300g oda sıcaklığında 1 dakika süreyle plakaları santrifüjleyin. 25 serum orta Kültür hücreleri ° C 22 saat veya 18 ° C 1-2 d. </li><li> Her bir kuyunun 30 ml serum içeren kültür ortamı ekleyin. </li&gt; <li> 300g oda sıcaklığında 1 dakika süreyle plakaları santrifüjleyin. </li><li> ~ 7 gün 25 nemli bir ek 5 odası ve kültürü birincil hücreler ° C tabak koyun </li><li> Resim hücreleri ya canlı ya da immünofloresan boyama sonra. </li></ol> 6. Temsilcisi Sonuçlar: En uygun koşullarda, kültür hücreleri yuvarlak bir morfoloji sağlıklı görünecektir. Kültür küçük hücre artıkları ve ilk kaplama sonra 50 ~% 80 birleşmiş olacak. Bu hücreler, uzun bir süre için kültür sonra morfolojik değişikliklere uğrayacaktır. Genellikle tam 25 farklı kültür hücreleri için yaklaşık 5-8 gün sürer ° C İyi kalite primer kültürlerinde genellikle hücrelerin-faiz kültürü ne kadar iyi ayırt tarafından değerlendirilecektir. Örneğin, birincil kas hücreleri genellikle bir dal gibi morfoloji ve multinükleer myotubes myofibrils gibi bir dizi sarcomeres oluşan şerit haline. Tamamen farklılaşmış myotubes kültüründe genellikle kendiliğinden hareket eder. Buna karşılık, farklı birincil nöronların hücre gövdeleri ile kümeler oluştururlar ve akson kabloları ile diğer kümeler ile bağlantı. <img src="/files/ftp_upload/2215/2215fig1.jpg" alt="Şekil 1" /> Şekil 1. Birincil hücre kültür kaplama sonra morfolojik değişikliklere uğrarlar Birincil hücreler (A) 384 plaka iyi sağ kaplama edildikten sonra parlak bir alan görüntü. Hücrelerin büyük çoğunluğu yuvarlak ve sağlam ve çok iyi ayrılmış. (B) Primer hücre kültürü parlak bir alan görüntü kaplama 20 saat sonra. İlköğretim hücreleri zaten bir şube gibi morfolojisi (uzun ok) kümeleri (büyük ok ucu) ve kaslar bu aşamada kabul edilebilir oluşturur. (C) primer kültür kaplama 5 gün sonra faz-kontrast görüntü. Kültür nöronal uzantıları (küçük oklar), nöron kümeleri (büyük ok başları) ve kasları (orta ölçekli ok), çok daha gelişmiş görünüyor. <img src="/files/ftp_upload/2215/2215fig2.jpg" alt="Şekil 2" /> Şekil 2. 384 plaka kültür dsRNAs ile tedavi birincil hücre örnekleri İlköğretim Dmef2-Gal4 taşıyan embriyolar, D42-Gal4 UAS-mito-GFP transgenlerin, hangi Dmef2 Gal4 ve kasları ve motor nöronlar mito-GFP D42-Gal4 sürücü ifadesi, sırasıyla hücreleri izole edildi. Hücreler lacZ (AC) veya şişirilmiş (eğer varsa) (DF) ya da hedef dsRNAs ile tedavi edildi. Birincil kas ve nöronların her ikisi de, GFP (A, C, D, F) tarafından görülebilir. Nöronal uzantıları beyaz oklar. Aktin (ok başları) Phalloidin boyama, kültür lacZ dsRNAs (B ve C) ile tedavi edilen bir şube gibi kas yapısı ortaya, ancak kültür yuvarlak kas morfoloji eğer tedavi dsRNAs (E ve F). Birleştirilen görüntülerin (C, F), kasları sarı lekeli, ama kırmızı negatif gösterir nöronlar ve onların uzantıları (beyaz oklar). <img src="/files/ftp_upload/2215/2215fig3.jpg" alt="Şekil 3" /> Şekil 3. Insan vitronectin kaplı kapak cam slaytlar kültüre birincil kasların Konfokal mikrograflar İlköğretim kasları immunofluorescently anti-Drosophila Titin (KZ) (birleştirme kırmızı) ve aktin için Phalloidin (birleştirme yeşil) ile boyandı. Üst paneller, vahşi-tip kontrol kas ve alt olanlar sals (CG31374) kısaltılmış sarcomeres RNAi kas vardır.

<ol>
	<li>Ashburner, M., Roote, J., Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory culture of Drosophila. , 588-599 (1999).</li><li>Bai, J., Hartwig, J. H., Perrimon, N. S. A. L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+WH2-domain-containing+protein,+promotes+sarcomeric+actin+filament+elongation+from+pointed+ends+during+Drosophila+muscle+growth.">a WH2-domain-containing protein, promotes sarcomeric actin filament elongation from pointed ends during Drosophila muscle growth.</a> Dev. Cell. 13, 828-842 (2007).</li><li>Bai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+screening+in+Drosophila+primary+cells+for+genes+involved+in+muscle+assembly+and+maintenance+.">RNA interference screening in Drosophila primary cells for genes involved in muscle assembly and maintenance .</a> Development. 135, 1439-1449 (2008).</li><li>Sepp, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+neural+outgrowth+genes+using+genome-wide+RNAi.">Identification of neural outgrowth genes using genome-wide RNAi.</a> PloS. Genet. 4, e1000111-e1000111 (2008).</li><li>Donady, J. J., Fyrberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+culturing+of+Drosophila+embryonic+cells+in+vitro.">Mass culturing of Drosophila embryonic cells in vitro.</a> TCA Manual. 3, 685-687 (1977).</li><li>Seecof, R. L., Alleaume, N., Teplitz, R. L., Gerson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+neurons+and+myocytes+in+cell+cultures+made+from+Drosophila+gastrulae.">Differentiation of neurons and myocytes in cell cultures made from Drosophila gastrulae.</a> Exp. Cell Res. 69, 161-173 (1971).</li><li>Bai, J., Sepp, K. J., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+Drosophila+primary+cells+dissociated+from+gastrula+embryos+and+their+use+in+RNAi+screening.">Culture of Drosophila primary cells dissociated from gastrula embryos and their use in RNAi screening.</a> Nature Protocols. 4, 1502-1512 (2009).</li></ol>

Drosophila hücrelerin primer kültürlerinin gelişim model, büyük ölçüde muhtemelen birincil hücre hazırlık sürecinde çeşitli değişkenlere bağlı olarak değişebilir . Her şeyden önce, yumurtlama için kullanılan uçar genç (eski bir hafta içinde) ve sağlıklı (viral ya da bakteriyel enfeksiyon ücretsiz). Bunlar işe yaramaz ve bu embriyoların elde edilen hücrelerin ayırt edemez ve sadece 1-2 gün hayatta olarak gübresiz veya enfekte embriyolar kaçınılmalıdır. İkincisi, homoje...

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<td>Shields and Sang M3 medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3652</td>
<td>&nbsp;</td>
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<td>Fetal bovine serum</td>
<td>&nbsp;</td>
<td>JRH Biosciences</td>
<td>12103-500M</td>
<td>&nbsp;</td>
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<td>Insulin from bovine pancreas</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I-6634</td>
<td>&nbsp;</td>
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<td>Large embryo collection cages</td>
<td>&nbsp;</td>
<td>Genesee Scientific</td>
<td>59-101</td>
<td>&nbsp;</td>
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<td>#25 US standard testing sieve</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>57334-454</td>
<td>&nbsp;</td>
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<td>#45 US standard testing sieve</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>57334-462</td>
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<td>#120 US standard testing sieve</td>
<td>&nbsp;</td>
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<td>57334-474</td>
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<td>Dounce tissue grinder, Wheaton 7 mL</td>
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<td>VWR</td>
<td>62400-620</td>
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<td>Dounce tissue grinder,Kontes 40 mL</td>
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<td>Dounce tissue grinder,Kontes 40 mL</td>
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<td>Costar, 384-well plate</td>
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<td>Lab-Tek II Chambered coverglass</td>
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<td>Fisher Scientific</td>
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primary cell cultures from drosophila gastrula embryos

Burada Drosophila gastrula embriyolar ayrı hazırlanması ve kültür birincil hücreler için bir yöntem açıklanmaktadır. Kısacası, büyük bir miktarı, genç ve sağlıklı bir sinek embriyolar sahnelenen, toplanan sterilize ve sonra bir cam Homojenizatör kullanarak tek bir hücre süspansiyonu içine fiziksel olarak ayrışmış. Kültür plakaları veya kültür ortamı uygun bir yoğunlukta odasına slaytlar kaplama olduktan sonra, bu hücreler daha fazla kendi özel hücre belirteçleri ile tespit edilebilir çeşitli morfolojik farklı hücre tipleri, ayırt edebilirsiniz. Ayrıca, çift zincirli (ds) RNA&#39;lar gen demonte ortaya çıkarmak için bu hücrelerin tedavisi için şartlar mevcut. Drosophila primer hücrelerde Verimli RNAi dsRNA içeren kültür ortamında hücrelerin sadece banyo yapılır. Diğer moleküler, biyokimyasal, hücre görüntüleme analizleri ile birlikte, etkili RNAi pertürbasyon yürütmek için yeteneği, özellikle Drosophila birincil hücreler, farklılaşmış kas ve nöronal hücrelerin ilgili olanlar olarak cevaplandırılmak üzere çeşitli sorular sağlayacaktır.

Watch this Scientific Journal Video about Primary Cell Cultures from Drosophila Gastrula Embryos at JoVE.com

Primary Cell Cultures from Drosophila Gastrula Embryos

Biz ayrı primer hücrelerin hazırlanması için ayrıntılı bir protokol<em&gt; Drosophil</em&gt; Embriyolar. Yeteneği, biyokimyasal, moleküler ve hücre görüntüleme yöntemleri ile birlikte, etkili RNAi pertürbasyon yürütmek için çeşitli sorular ele alınacak izin<em&gt; Drosophila</em&gt; Birincil hücreler.

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 ...

primary-cell-cultures-from-drosophila-gastrula-embryos

Research

JoVE Journal

Biology

Primary Cell Cultures from Drosophila Gastrula Embryos

13.8K Görüntüleme.  Harvard Medical School. Biz ayrı primer hücrelerin hazırlanması için ayrıntılı bir protokol Drosophil Embriyolar. Yeteneği, biyokimyasal, moleküler ve hücre görüntüleme yöntemleri ile birlikte, etkili RNAi pertürbasyon yürütmek için çeşitli sorular ele alınacak izin Drosophila Birincil hücreler.

Video: Primary Cell Cultures from Drosophila Gastrula Embryos

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

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 ...

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 ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

Doku Hedeflenen Embriyonik Chimeras: Zebra balığı Gastrula Hücre Nakli

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

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 ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

VE Dik Görüntüleme Drosophila Embriyolar

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Living Görüntüleme Hücre Şekli Değiştir Drosophila Embriyolar

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Kinesin İzolasyonu ve Saflaştırılması gelen Drosophila Embriyolar

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

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&#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 ...