Мы хотели бы поблагодарить Томаса Lecuit (SQH-атрофина :: GFP линия), в Блумингтон со-центр, и процесс развития Studies гибридомные банк для реагентов. Кроме того, мы благодарим всех членов Tootle Lab за полезные обсуждения и критики рукописи. Финансирование от Национального научного фонда MCB-1158527, и исходные финансовые средства от анатомии и Департамента клеточной биологии, Университет Айовы поддержала эту работу. Национальные институты здравоохранения Predoctoral подготовки Грант в фармакологических наук T32GM067795 поддерживается AJS. Поддержка хранения данных была предоставлена ​​ИКТ, который финансируется через CTSA при поддержке Национального Центра Ресурсов Исследования и Национального центра Продвижение поступательного науки, Национальные Институты Здоровья, через Грант UL1RR024979.

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Там нет ничего, чтобы раскрыть.

Drosophila фолликул состоит только из небольшого числа типов клеток, что делает его идеально подходит как для морфологических и молекулярных анализов. Кроме того, из-за структуры яичника, это относительно легко получить большое количество конкретных стадиях развития фолликулов с общ?...

Каждый дрозофилы яичников состоит из ~ 16 овариол или цепочек последовательно созревания яиц камер или фолликулов. Каждый фолликул состоит из одного ооцита, 15 зародышевой линии, полученные медсестра или поддерживающих клеток и ~ 650 соматических клеток, называемых фолликул клеток (фиг. 1A). Drosophila оогенез разделена на 14 морфологически определенных стадиях развития 1. Каждый этап развития фолликула наблюдается много раз в пределах одного лету, что делает его относительно легко изолировать значительное количество сценических конкретных фолликулов. В середине-конце этапы оогенезе (Этапы 10B-14) особенно хорошо подходят для изоляции стадии (рис. 1). В Этап 10B (S10B), фолликул полностью вытянута (т.е. его длина равна стадии 14 (S14) фолликула, см. рисунок 1 и рисунок 2H) и половины длины фолликула состоит из питающих клетокв то время как другая половина является ооцитов (рис. 1С). На данном этапе медсестра клетки подвергаются драматический актина ремонт, укрепление корковой актин и генерации параллельных пучков нитей актина 2. В то же время, население ячейки стручка, называется центростремительные клетки, мигрируют в между питающих клеток и яйцеклетки, и два спинных группы ячейки стручка стать указанный пройти миграции для формирования спинные придатки, трубчатые дыхательные аппараты для эмбриона 3 . Медсестра клетки затем контракт (S11), сжимая свои цитоплазматические содержимое в яйцеклетку в процессе, называемом медсестра клеток демпинга, который обеспечивает яйцеклетки с виду факторы, необходимые для того, чтобы завершить эмбриогенеза (рис. 1D). Медсестра клетки затем пройти гибель клеток (S12-S13) 4, и фолликул клетки секретируют и рисунок на яичную скорлупу 5 (рис. 1E-G). Таким образом, конец оогенезе богат важной ап развитияг морфогенетические процессы. Изолированные середине-конце этапа фолликулы (S10B-S14) может быть использован для различных целей, в том числе молекулярных анализов. Например, мРНК из фолликулов, организованных могут быть выделены для ОТ-ПЦР, микрочипов, или РНК-SEQ анализов. Это позволяет смотреть на экспрессии генов в течение короткого окна развития, с помощью всего нескольких типов клеток, присутствующих, и определить, как экспрессия генов изменяется либо фармакологических или генетических возмущений. Стадия изоляция может также использоваться, чтобы смотреть на белках с помощью вестерн-блоттинга. Такой анализ важен, поскольку он позволяет количественно уровень экспрессии белка дикого типа по сравнению с мутантами на определенных этапах. Хотя можно было бы использовать иммунофлуоресцентного анализа для достижения аналогичных результатов, количественное определение флуоресценции менее надежными из-за строгих требований, которые все пиксели быть в пределах линейного диапазона обнаружения 6. Кроме того, вестерн-блот анализ может предоставить другую информацию, напримерс если белок посттрансляционно изменены или выражается с определенной изоформы сплайсинга. Изолированные этапы также могут быть использованы для дальнейшей очистки белка, в том числе внутриклеточного фракционирования или коиммунопреципитации подтверждено. Стадия конкретных изоляции фолликул также может быть использован для в пробирке анализов развития 7 и живого изображения 8. Изолированные S10B-S13 фолликулы будут и впредь развиваться на S14 в простой питательных сред (см. ниже). Важно отметить, что S10A фолликулы не будет прогрессировать через медсестры клетки сброс с использованием условий культивирования, обсуждаемые в этой рукописи. Мы использовали S10B в пробирке анализов развития определить роль простагландинов, как фармакологически и генетически, в регулировании актина ремоделирования с помощью медсестры ячейку демпинг и развитие как прочитанные-аутов 7,9. Точно так же более поздних стадиях развития могут быть также выделены для определения последствий фармакологического лечения или генетической человекаipulations по конкретным процессов, таких как центростремительной миграции клеток, спинной придатком миграции / формирования 10, и смерти медсестры клеток. Такие анализы могут быть использованы для выполнения доминирующие экраны взаимодействия или анализов, например, в то время как гетерозиготность мутаций в PXT или fascin только не имеют никакого влияния на S10B развития в пробирке, фолликулы от двойных гетерозигот экспонат медсестра клеток демпинговым дефекты и блок в развития 9. Кроме того, поскольку S10B-13 может развиваться в культуре, все процессы, которые происходят в это время можно наблюдать живой-изображений. Такая процедура может быть проведена просто с помощью проходящего света (если интересоваться только в грубых изменений в морфологии) или с конфокальной микроскопии с использованием трансгенных мух, выражающие флуоресцентных зондов или фолликулов, окрашенные в прямом эфире изображений красителей. Онлайн изображений используется существенно продвинуть наше понимание процессов развития. Действительно, жить Imaginг поздней стадии фолликулов расширила знания спинной придаток миграции, пример тубулогенеза 10. Мы ожидаем, что жить изображений дополнительных поздней стадии процессов, в том числе динамики актина во время медсестра клетки демпинговым, обеспечит новые идеи этих событий в развитии. Важно отметить, что в то время как S10A и ранние этапы развития фолликула не будет продолжать развиваться в S14 в культуре, жить-визуализации событий, происходящих в течение этих стадиях развития возможно с помощью альтернативных условиях культивирования 11-14 (см. Обсуждение для более информация). Здесь мы предлагаем подробные протоколы для изоляции поздней стадии фолликулы либо развития в пробирке и жить-визуализации или молекулярного анализа (мРНК и изоляции белка).

<table border="0" cellpadding="0" cellspacing="0" width="1098">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:253px;">Active Dry Yeast</td>
			<td style="width:195px;">Genesee Scientific</td>
			<td style="width:227px;">62-103</td>
			<td style="width:424px;">Any source of Active Dry Yeast is fine</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Grace&#8217;s Insect Media</td>
			<td>Lonza</td>
			<td>04-457F</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Heat Inactivated Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11050H</td>
			<td>Any Heat Inactivated FBS should work</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">10x Pen/Strep</td>
			<td>Gibco/Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pin Vises and Needles</td>
			<td>Ted Pella, Inc.</td>
			<td>13561-10</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Spot Plate, Nine Well</td>
			<td>Corning</td>
			<td>7220-85</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">#5 Dumont forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">24 multi-well plates</td>
			<td>Becton Dickinson</td>
			<td>35 3226</td>
			<td>Any 24-well tissue culture dish should work</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Coverslip Bottom Dishes (35mm)</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.0-14-C</td>
			<td>Coverslip thickness will depend on the microscope/objective being used</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glass pipettes</td>
			<td>Corning</td>
			<td colspan="2">7095B-5x (for transferring follicles)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glass pipettes - long</td>
			<td>Corning</td>
			<td colspan="2">7095B-9 (for producing pulled pipettes)</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">Sample pestle (1.5 &#956;l; RNase/DNase free)</td>
			<td>Research Products International</td>
			<td>199228</td>
			<td>Any plastic pestle that fits 1.5 &#956;l microfuge tubes can be used</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Trizol</td>
			<td>Invitrogen</td>
			<td>15596-018</td>
			<td></td>
		</tr>
	</tbody>
</table>

the utility of stage-specific mid-to-late drosophila follicle isolation

Drosophila оогенез или развитие фолликула широко используется для продвижения понимания сложных развития и клеточных биологических процессов. Это методы статья описывает, как изолировать середине-конце этапа фолликулы (Стадия 10В-14) и использовать их, чтобы обеспечить новому взглянуть на молекулярном и морфологических событий, происходящих во время жесткой окон времени развития. Изолированные фолликулы могут быть использованы для различных экспериментальных методов, в том числе в пробирке анализов развития, живого изображения, анализа экспрессии мРНК и Вестерн-блоттинга белков. Фолликулы на стадии 10B (S10B) или более поздней версии завершит разработку в области культуры; это позволяет объединить генетические или фармакологические возмущения с развитием в пробирке, чтобы определить последствия таких манипуляций на процессы, происходящие в определенные периоды развития. Кроме того, поскольку эти фолликулы развиваются в культуре, они идеально подходят для изучения живого изображения, Которые часто показывают новые механизмы, которые опосредуют морфологические события. Изолированные фолликулы также могут быть использованы для молекулярных анализов. Например, изменения в экспрессии генов, которые являются результатом генетических возмущений может быть определен для конкретных окон развития. Кроме того, уровень белка, стабильность, и / или посттрансляционная состояние модификация в течение определенного этапа развития фолликула может быть рассмотрено через вестерн-блот анализа. Таким образом, изоляция стадиеспецифический из Drosophila фолликулов обеспечивает богатый источник информации в широко консервативных процессов развития и морфогенеза.

Когда выделения специфических этапы развития Drosophila фолликула важно иметь возможность точно различать различные морфологические этапы. Это несколько сложным для S10A и S10B, как медсестра клетки и ооцит каждый занимают половину длины фолликула на этих этапах (Фиг.1В по сравн?...

Watch this Scientific Journal Video about The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation at JoVE.com

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

Стадия конкретных изоляция середине-конце Drosophila фолликулов полезна для различных целей. Такие фолликулы развиваются в культуре, что позволяет генетических и / или фармакологических манипуляций, чтобы использоваться в сочетании с в пробирке анализов развития и живого изображения. Кроме того, фолликулы могут быть использованы для молекулярных исследований, таких как изолирующий мРНК и белок.

Утилита Стадия конкретных Середина-конце<em&gt; Дрозофилы</em&gt; Фолликул Изоляция

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

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

11.2K Views.  University of Iowa Carver College of Medicine.  Стадия конкретных изоляция середине-конце Drosophila фолликулов полезна для различных целей. Такие фолликулы развиваются в культуре, что позволяет генетических и / или фармакологических манипуляций, чтобы использоваться в сочетании с в пробирке анализ...

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

Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft during early anagen, the growth phase of the hair cycle, as well as pluripotent stem cells that play a role in hair follicle growth but have the potential to differentiate to non-follicle cells such as neurons. These properties of the hair follicle are discussed. The various cell types of the hair follicle are potential targets for gene therapy. Gene delivery system for the hair follicle using viral vectors or liposomes for gene targeting to the various cell types in the hair follicle and the results obtained are also discussed.

The hair follicle is a highly complex appendage of the skin containing a multiplicity of cell types. The follicle undergoes constant cycling through the life of the organism including growth and resorption with growth dependent on specific stem cells. The targeting of the follicle by genes and stem cells to change its properties, in particular, the nature of the hair shaft is, discussed. Hair follicle delivery systems are described, such as liposomes and viral vectors for gene therapy. The nature of the hair follicle stem cells is discussed, in particular, its pluripotency.

Целевые Экспрессия GFP в волосяной фолликул Использование Ex Vivo Вирусный трансдукция

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

Флуоресцентная маркировка Drosophila Структур сердца

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

Визуализация бьющееся сердце в Drosophila</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Выделение и очистка Drosophila Периферийные Нейроны магнитными бисера Сортировка

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

Изоляция Дрозофилы Семенники

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

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.

Выделение и очистка от кинезин Drosophila Эмбрионы

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

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Живая съемка апоптоза клеток во время акции<em&gt; Drosophila</em&gt; Эмбриогенеза

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

Affinity основе Выделение Tagged ядер от<em&gt; Дрозофилы</em&gt; Ткани для анализа экспрессии генов

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

Isolation of Neonatal Extrahepatic Cholangiocytes

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood.
Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established1-4. Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts.
This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.

A technique to isolate cholangiocytes from the extrahepatic bile ducts of neonatal mice is described. The ducts are meticulously dissected, and then cells are isolated by outgrowth in thick collagen gels. This method provides a useful tool for studying extrahepatic bile duct development and pathology.

Выделение новорожденных внепеченочных холангиоцитах

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Выделение Мышиный клапана эндотелиальных клеток

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...