Biz hastalara bu deneyler dokusu toplamalarında onların katılım için borçlu bulunmaktadır. Bir DoD OCRP yumurtalık kanseri Akademi Ödülü (W81XWH-15-0253) ve Anirban K. Mitra için Colleen'in rüya Vakfı Ödülü pilot bu araştırma desteklenen.

Protokol kurumsal düzenleme kurulu, Indiana Üniversitesi kuralları izler.
1. hücre hazırlık
<ol><li>
Yalıtım ve kültür insan birincil mesothelial hücreler
	<ol>
<li>Ayrı tutma insan birincil mesothelial hücreleri (HPMCs)17,18 daha önce açıklanan ve onları büyümek insan omentumun büyüme orta [Dulbecco'nın modifiye kartal Orta (% 10 fetal Sığır serum, % 1'i içeren DMEM) tamamlamak penisilin-streptomisin, % 1 esansiyel olmayan amino asitler ve % 1'i vitamin] 37 ° C ve % 5 CO2. Not: HPMCs genellikle % 100 izdiham (Şekil 1A) yetiştirilmektedir.</li>
	</ol>
</li>
	<li>
Yumurtalık kanser hücrelerinin büyüme koşulları
	<ol>
<li>Tam büyüme orta 37 ° C ve % 5 CO2HeyA8 yumurtalık kanseri hücrelerinde büyür. Not: Kullanım HeyA8 yetiştirilen yaklaşık % 80'i izdiham (Şekil 1B) hücreleri.</li>
	</ol>
</li>
</ol>2. hücre kültürü
<ol><li>
Proksimal kültür set-up
	<ol>
<li>6-iyi plakaları ile 10 mikron kalınlığında Polikarbonat doku kültürü tedavi membran 0.4 mikron gözenekleri (108 gözenekleri/cm2) ve 4,67 cm2 büyüme alanı için kullanım Transwell ekler. Gerekirse, optimize etmek için farklı Ekle boyutları göre büyüme yüzey alanı seribaşı hücre sayısı ölçekleyerek. Sıcak tam büyüme orta, tripsin ve fosfat tamponlu tuz (PBS) kullanmadan önce 37 ° c.</li>
	</ol>
</li>
	<li>
Ekler hazırlanıyor
	<ol>
<li>HeyA8 hücre membran ucun alt yüzeyinde tohum için INSERT steril forseps kullanarak onun paketinden çıkarın ve bir steril 15 cm kültür tabak içinde ters yerleştirin.</li>
		<li>Derinliği ters Ekle karşılamak ya da herhangi bir uygun Steril konteyner ile vekil için olduğu gibi 15 cm tabak kullanın. Deney bağlı olarak ekler gerekli sayıda 15 cm çanak yerleştirin.</li>
		<li>Buna göre üç denetimi ve üç deneysel koşullar etiket ekler kenarında bir marker ile.</li>
	</ol>
</li>
	<li>
Kanser hücrelerinin hazırlanıyor
	<ol>
<li>25 cm2 şişeye (~ 2 x 106 hücreler) % 80 izdiham, yetiştirilen HeyA8 hücre trypsinize, 40 – 60 s için 1 mL tripsin (%0.25) ekleyin ve tripsin tam büyüme ortamının 6 mL ile nötralize etmek için.</li>
		<li>500 x g oda sıcaklığında (RT) 3 dk de hücreleri santrifüj kapasitesi, süpernatant atmak ve hücre Pelet tam büyüme orta 5 mL resuspend. Trypan mavi boya hariç tutma yöntemini kullanarak canlı hücreleri saymak.</li>
	</ol>
</li>
	<li>
Kanser hücrelerinin tohumlama
	<ol>
<li>100.000 hücre/mL tam büyüme orta canlı HeyA8 hücrelerinin askıya alma. Tam büyüme orta (ters beri bu şimdi, karşı karşıya) Ekle alt 800 μL içinde dikkatle tohum 80.000 HeyA8 hücreleri.</li>
		<li>Böylece hücreleri Ekle (Şekil 1 c) kalır bir kubbe gibi şekli oluşturmak için hücre tohum. Membran Merkezi'nden başlayın ve yavaş yavaş pipetting süre eşmerkezli daireler dışa doğru hareket. 3 mm den fazla herhangi bir orta dökülmesini önlemek için kenarından gidiyor kaçının. Not: Numaralı seribaşı hücre sayısı hücre büyüme oranı ve deneme süresi bağlıdır. Proksimal bu kültürü için tohumlari HeyA8 hücre sayısı optimize ve hücreleri üçüncü günün sonunda % 80 – 90 birleşmesi olacak.</li>
	</ol>
</li>
	<li>
Kanser hücre Eki
	<ol>
<li>15 cm çanak kapağı ve dikkatle CO2 kuluçka için ekler içeren yemek taşımak. Orta ve hücreleri üzerine yerleştirin Bu adımda damla bozmaya değil önemlidir. Kanser hücrelerinin Ekle'nin üzerine takmak izin vermek 4 h için 37 ° C'de hücreler bırakın.</li>
	</ol>
</li>
	<li>
Mesothelial hücreler hazırlanıyor
	<ol>
<li>Yaklaşık 4 saat sonra % 100 izdiham tripsin (% 0,25) 1-2 min için 2 mL ile 75 cm2 şişeye (~ 4 x 106 hücreler) içinde yetiştirilen HPMCs trypsinize ve tripsin tam büyüme ortamının 12 mL ile nötralize.</li>
		<li>Hücreleri, 500 x g de RT 3 dk santrifüj kapasitesi, hücre Pelet 10 mL tam büyüme orta resuspend ve trypan mavi boya hariç tutma yöntemini kullanarak canlı hücreleri saymak.</li>
	</ol>
</li>
	<li>
Mesothelial hücreler tohumlama
	<ol>
<li>Canlı HPMCs 300.000 hücre/mL tam büyüme aracı olarak askıya alma. Taze tam büyüme ortamının 2.5 mL 6-şey plaka her şey için ekleyin. Dikkatle Biyogüvenlik hood için dışarı ekler içeren 15 cm çanak getir. Steril forseps kullanarak, INSERT flip ve dik, tam büyüme orta (Şekil 1 c) dalmış alt yüzey bağlı HeyA8 hücreleri ile 6-şey kalıbının kuyuya yerleştirin.</li>
		<li>Tohum 450.000 HPMCs (Şekil 1 c) kuyunun bakan yüzeyi veya ekler olmadan herhangi bir HeyA8 hücreleri HPMC denetimler için bağlı HeyA8 hücreler ekler iç büyüme ortamının 1,5 ml askıya. Hücreleri olmadan büyüme ortamının 1.5 mL HeyA8 denetimleri ekleyin.</li>
	</ol>
</li>
	<li>
Proksimal kültür büyüyen
	<ol>
<li>Proksimal kültür için 72 h 37 ° C ve %5 CO2 CO2 kuluçka büyümeye izin. Gerekirse, orta aşağıdaki gibi doldurulan.<ol>
<li>İç ekleme için 750 µL kaldırın ve taze tam büyüme ortamının 750 µL ekleyin.</li>
			<li>Dış ekleme (6-şey plaka) için 1 mL kaldırın ve 1 mL taze tam büyüme ortamının ekleyin. 1 mL tek adımda orta değiştirme kolaylığı için seçildi. İsterseniz, 1,25 mL kaldırıldı ve yerine.</li>
		</ol>
</li>
	</ol>
</li>
	<li>
Hücre toplama
	<ol>
<li>Hücreleri proksimal kültür 72 h sonra toplamak. Hücreleri çapraz bulaşma HeyA8 hücreleri ve HPMCs. Trypsinize önlemek için bu adımı, özel dikkat, onları santrifüj kapasitesi ve çapraz kontaminasyonu önlemek için membran hücrelerdeyse lysing yerine Pelet parçalayıcı.</li>
	</ol>
</li>
	<li>
Hücreleri trypsinizing
	<ol>
<li>Her iki ucun 1 x PBS ile yıkayın.</li>
		<li>Yeni bir de ekler nakletmek ve tripsin eklemek. Tripsin 0.5 mL ucun içeriye doğru ekleyin ve tripsin 2 mL 6-şey kalıbı Ekle alt yüzeyine bağlı kanser hücrelerinin trypsinize ekleyin. 37 ° C'de 2 dk plaka kuluçkaya</li>
	</ol>
</li>
	<li>
Tripsin nötralize
	<ol>
<li>Tam büyüme orta 1 mL tripsin nötralize, yukarı ve aşağı, pipet ve sonra hücreleri almak ve etiketli toplama tüp aktarmak için INSERT içine ekleyin. Tam büyüme ortamı tam bir nötralizasyon için bir ek 2 mL ekleyin. Membran değil delmek hücrelerin bir çapraz kontaminasyonu önlemek için pipetting süre için özen gösterilmelidir.</li>
		<li>Hücreleri ekleme alt toplamak için böylece hücreleri 6-şey kalenin dibine oldu ekledi adım 12,2 alt yüzeyine 2-3 x 2 mL tripsin pipette. Tripsin plaka altındaki büyüme ortamının 6 mL ile etkisiz hale getirmek ve etiketli toplama tüp plaka içeriğini aktarmak.</li>
	</ol>
</li>
	<li>
Toplama ve hücre lysing
	<ol>
<li>500 x g 3 dk de hücreleri santrifüj kapasitesi ve medya Aspire edin. Resuspend ve fenol ve guanidin thiocyanate-tabanlı lizis reaktif RNA çıkarılması için 0.7 mL ile hücre topakları parçalayıcı. Herhangi bir uygun kiti kullanarak RNA yalıtmak.</li>
	</ol>
</li>
</ol>3. kantitatif gerçek zamanlı polimeraz zincir reaksiyonu
Not: Aşağıdaki adımlarda bir nicel gerçek zamanlı polimeraz zincir gen ifadeleri değişikliklerin sonucu olarak proksimal kültür çalışmaya tepkimesi (qPCR) açıklanmıştır.
<ol><li>CDNA RNA örneklerinden qPCR için hazır olun. Uygun herhangi bir ters transkriptaz Ters transkripsiyon tüm mRNA emin olmak için karşılık gelen bir tampon ve rasgele astar ile kullanın (bkz. Tablo malzeme).</li>
	<li>Fibronektin (FN1) ve E-cadherin (CDH1) mRNA ifade düzeyde değişiklikleri değerlendirme ve büyüme faktörü beta 1 (TGFβ1) tarafından qPCR gliseraldehit 3-fosfat dehidrogenaz (GAPDH) olarak bir iç kontrol ile dönüşümü. Standart ticari olarak mevcut gen ifade deneyleri ( Tablo malzemelerigörmek) kullanmak veya astar tasarım için mümkündür.</li>
	<li>HeyA8 kontrolleri ile HPMCs ve proksimal kültür HPMC kontrolleri ile yetiştirilen HPMCs ile proksimal kültüründe yetiştirilen HeyA8 hücreleri karşılaştırın.</li>
</ol>

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Yazarlar ifşa gerek yok.

Parakrin ve juxtacrine hücreler arasında sinyal mekanizmasının anlama, normal doku homeostazı hastalığı koşullar7,ve8hakkında daha iyi bilgi geliştirmek için önemlidir. Çoğu parakrin toplayarak yürütülen çalışmalar sinyal orta bir hücre tipi ve diğer hücre tipi tedavi etmek için kullanarak şartına. Bu yöntem doğal sadeliği içinde bir avantaja sahiptir. Ancak, bu doğru hücresel microenvironment salgılanan faktörler yerelleştiril...

Kanser hücreleri ve tümör microenvironment tümör ilerleme arasındaki üretken karşılıklı etkileşimler rolü de kurulmuş olup araştırma kanser biyoloji1' deki önemli bir odak noktası haline gelmiştir. Çift yönlü sinyal benzer örnekleri şifa, bağışıklık yanıtı, anjiogenez, kök hücre nişler, yara ve geliştirme2,3,4,5,6 sırasında açısından büyük önem taşıyor , 7 , 8. hücreleri hücre kaderi, doku fizyolojisi ve hastalık ilerleme belirleyen çeşitli şekillerde ekstraselüler ipuçlarını kendi microenvironment gelen tepki bu biyolojik süreçler içinde ortak bir tema olduğunu. Bu nedenle, odak giderek tür hücre-hücre iletişim içinde yer alan mekanizmaları daha iyi bir anlayış geliştirmek doğru döndü. Bu tür etkileşimler çoğunluğu ücretli olabilecek parakrin veya hücreler arasında sinyal juxtacrine. Parakrin sinyal bir yanıtta ise9,10, tetikleme çevresinde, tabloda başka bir hücreyi tarafından karşılık gelen reseptörleri juxtacrine algılanan spesifik sinyal faktörler bir hücre salgılanmasını içerir sinyal iki hücre dahil11,12hücresel bileşenleri arasında doğrudan temas gerektirir.
Böyle sinyal doku homeostazı, hem de tümör microenvironment çok önemli bir bileşenidir. Kanser hücrelerinin parakrin ve juxtacrine faktörleri kanser ilişkili fibroblastlar (restorantlar), bağışıklık hücreleri ve adiposit13,14,15,16gibi tümör stroma hücreden faydalanın. Juxtacrine sinyal bitişik ligandlar ve çentik sinyal veya integrinler ve onların anılan sıraya göre arasındaki etkileşimler reseptörler içerir iken büyüme faktörleri, sitokinler, kemokinler, vb, aracılı sinyal parakrin hücre dışı matriks proteinleri. Yumurtalık kanseri hücreleri ve restorantlar arasındaki karşılıklı etkileşimler tümör ilerleme ve metastaz14önemini göstermiştir. Benzer şekilde, anahtar mikroRNA ve metastatik kolonizasyon17, teşvik kanser hücreleri transkripsiyon faktörleri Onkolojisti yumurtalık kanser hücrelerinin metastaz sitenin kapsayan mesothelial hücreler ile etkileşimleri düzenleyen 18.
Bir şartına orta ikinci hücre tipi ile tedavi etmek için bir hücre türünden toplanan kullanımı dahil parakrin sinyal üzerinde çoğu çalışmaları. Bu yaklaşım yaygın olarak kullanılan iken, alan hücre microenvironment salgılanan faktöründeki yerelleştirilmiş yüksek konsantrasyon düzeyleri etkili çoğaltmaz. Aynı zamanda bir hücre tarafından üretilen salgılanan faktörü, sürekli akış kinetik çoğaltmak başarısız olur ve komşu hücre tarafından alınan. Parakrin salgılanan faktörler sadece kaynak hücre çevresinde gerekli konsantrasyonlarda ve diffüz ve mesafe arttıkça dışarı seyreltik eğilimi gibi kısa mesafelerde etkilidir sinyal. Salgılanan faktör bu yerelleştirilmiş yüksek yoğunlukta bir yanıt reseptör hücre olarak tetiklemek için esastır. Ayrıca, alıcı hücrelerdeki yanıt da yeni salgılanan faktörler ve onların sürekli tükenmesi bozulması, bağlama ve içselleştirilmesi alıcı hücreler ve kaynak hücreye uzak difüzyon yoluyla denge bağlıdır. Klimalı orta yüksek yerelleştirilmiş konsantrasyonları microenvironment içinde mevcut için hesabına konsantre olabilir, ama bu doğru bir şekilde tam konsantrasyonları çoğaltma yapamaz. Ayrıca, üretimin doğal kinetik ve faktör ilgili tükenmesi taklit edemez. Daha doğrusu parakrin sinyal çoğaltmak ve mekanizmaları sinyal juxtacrine ayırmak için her iki yüzeyi geçirgen membran iki hücre tipleri büyüyen içerir bir roman proksimal kültür yöntemi tasarladılar. Gözenekleri juxtacrine etkileşimleri önlemek ve henüz yerelleştirilmiş yüksek konsantrasyonları salgılanan faktörler değişimine izin vermek için küçük. Bu şekilde, bu sistem üretim kinetik ve parakrin faktörler tükenmesi korur.

<table>
	<tbody>
		<tr>
			<td>24 mm Transwell permeable support with 0.4 &#181;m Pore Polycarbonate Membrane Insert</td>
			<td>Corning (Costar)</td>
			<td>3412</td>
			<td>&#8226; 10 &#181;m thick translucent polycarbonate membrane 
			&#8226; Treated for optimal cell attachment 
			&#8226; Packaged 6 inserts in a 6 well plate, 4 plates per case 
			&#8226; Membrane must be stained for cell visibility 
			&#8226; Sterilized by gamma radiation</td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Corning (Falcon)</td>
			<td>353046</td>
			<td>Flat Bottom, TC-treated, sterile, with Lid</td>
		</tr>
		<tr>
			<td>15 cm culture dish</td>
			<td>Corning (Falcon)</td>
			<td>353025</td>
			<td>Sterile, TC-treated Cell Culture Dish</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning (Cellgro)</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Nonessential amino acids</td>
			<td>Corning (Cellgro)</td>
			<td>25-025-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Vitamins</td>
			<td>Corning (Cellgro)</td>
			<td>25-020-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin, 2.21 mM EDTA</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipets</td>
			<td></td>
			<td></td>
			<td>Any make is fine</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td></td>
			<td></td>
			<td>Any make is fine</td>
		</tr>
		<tr>
			<td>Biosafety level II cabinet</td>
			<td></td>
			<td></td>
			<td>Any make is fine</td>
		</tr>
		<tr>
			<td>FN1 TaqMan Gene Expression Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs01549976_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>TGFB1 TaqMan Gene Expression Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs00998133_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CDH1 TaqMan Gene Expression Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs01023895_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH TaqMan Gene Expression Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs99999905_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>miRNeasy mini RNA isolation Kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>43-688-13</td>
			<td></td>
		</tr>
		<tr>
			<td>HeyA8 ovarian cancer cells</td>
			<td>Obtained from Ernst Lengyel Lab, University of Chicago</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TGF&#946; Neutralizing Antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB1835-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

a proximal culture method to study paracrine signaling between cells

Hücreler arası etkileşimler tümör ilerleme, bağışıklık yanıtı, anjiogenez ve geliştirme de dahil olmak üzere birçok biyolojik süreçlerinde önemli bir rol oynamaktadır. Parakrin veya juxtacrine sinyal bu tür etkileşimler aracılık eder. Bir şartına orta ve coculture çalışmalar etkileşimleri bu iki tür arasında ayırımcılık için en yaygın yöntem vardır. Ancak, yerelleştirilmiş yüksek konsantrasyonda microenvironment salgılanan faktörler parakrin etkileşimleri sırasında etkisini doğru tarafından şartına orta recapitulated değil ve böylece, imprecise sonuçlara yol açabilir. Bu sorunu çözmek için parakrin sinyal çalışmaya proksimal kültür yöntemi tasarladılar. İki hücre tipleri 0.4 µm gözenekli bir 10 µm kalınlığında Polikarbonat membran her iki yüzeyi yetiştirilmektedir. Gözenekleri salgılanan faktörler değişimine izin ver ve aynı zamanda, juxtacrine sinyal inhibe. Hücreleri toplanan ve uç noktada parakrin sinyal etkilerini belirlemek için lysed. Salgılanan faktörlerin yerelleştirilmiş konsantrasyon degradeler için izin vermeye ek olarak, bu yöntem takmaktan kültürünün yanı sıra inhibitörleri kullanımını içeren deneyler için mükellef olur. Biz yumurtalık kanseri hücreleri ve metastaz sitesinde karşılaştıkları mesothelial hücreler arasındaki etkileşimler eğitim için bu yöntemi kullanırken, çeşitli alanlarda sinyal parakrin çalışmaya araştırmacılar için herhangi bir iki yapışık hücre tipleri için adapte edilebilir, tümör microenvironment, immünoloji ve geliştirme de dahil olmak üzere.

Onkolojisti yumurtalık kanser hücrelerinin metastaz periton boşluğuna19içinde yerinde mesothelial hücreler karşılaşma. Mesothelial hücreler yardımıyla uyarlamalı başarılı metastaz17,18,20,21 etkinleştirmek yumurtalık kanser hücrelerinin yanıt-e doğru inducing içinde üretken parakrin ve juxtacrine etkileşimleri . Pr...

Watch this Scientific Journal Video about A Proximal Culture Method to Study Paracrine Signaling Between Cells at JoVE.com

A Proximal Culture Method to Study Paracrine Signaling Between Cells

Parakrin ve juxtacrine hücresel etkileşimlerin tümör ilerleme, bağışıklık yanıtı, anjiogenez ve geliştirme de dahil olmak üzere birçok biyolojik süreçlerinde önemli bir rol oynamaktadır. Burada, proksimal kültür yöntemi parakrin sinyal incelemek için doğrudan hücresel temas engelleyen sırasında salgılanan faktörler yerelleştirilmiş konsantrasyonları nerede saklanır kullanılır.

Parakrin hücreler arasında sinyal çalışmaya proksimal kültür yöntemi

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and ...

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Research

JoVE Journal

Biology

9.4K Görüntüleme.  Indiana University School of Medicine. Parakrin ve juxtacrine hücresel etkileşimlerin tümör ilerleme, bağışıklık yanıtı, anjiogenez ve geliştirme de dahil olmak üzere birçok biyolojik süreçlerinde önemli bir rol oynamaktadır. Burada, proksimal kültür yöntemi parakrin sinyal incelemek için doğrudan hücresel temas engelleyen sırasında salgılanan faktörler yerelleştirilmiş konsantrasyonları nerede saklanır kullanılır.

Video: A Proximal Culture Method to Study Paracrine Signaling Between Cells

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Kertenkeleler Hareket Hız ve Yön Visual Hassasiyet Test

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Biyoloji

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Erken Civciv Embriyolar ex vivo (Kültür) Kültür Yöntemi

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

S. cerevisiae &quot;adlı organel morfolojisi küresel çalışma yüksek verimli bir yöntem

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Hi-C: genomların Üç boyutlu Mimari Eğitim Bir Yöntem.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Newt Lens Rejenerasyon Eğitim Kültürleme İris Pigment Epitel Hücreler için Sistem

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Wild-tip ve Süs Çeşitler arasında ayırt A PCR tabanlı Genotiplendirme Yöntemi Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

Vasküler Ca Görselleştirme 2 + Parakrin Türetilmiş ROS tarafından tetiklenmiş Sinyal

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

İfade Klonlama tarafından Grb2 Sinyal Kompleksi Eğitim için bir Yüksek içerik Görüntüleme İş Akışı

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

Yeni Bir<em&gt; Ex vivo</emEmbriyonik Fare Kalp için&gt; Kültür Yöntemi

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Hücre Tek katmanlar mekanik uyarılması bağlı Kalsiyum Dalga Yayılım: Sığır Kornea Endotel Hücreleri Örneği

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...