This study was supported by a Merit Award from the U.S. Department of Veterans Affairs, and an NIH grant R01CA1385282522717 to RW.

1. Подготовка материалов и реагентов <ol><li> Этикетка каждую лунку культуре ткани обрабатывают 6-луночного планшета соответственно для каждой клеточной линии или состояния, которое исследованы. </li><li> Подготовьте 2x клеточную культуральную среду путем растворения 1 г порошка среды и 0,2 г бикарбоната натрия в деионизированной водой до конечного объема 50 мл. </li><li> Передайте эту среду через 0,2 мкм фильтр для стерилизации. </li><li> Добавить дополнительные компоненты, необходимые для нормальной культуры клеточной линии, представляющей интерес. Например, растут СМТ линию 167 клеток в RPMI 1640 среде, дополненной 10% FBS и 1% раствор пенициллина / стрептомицина. Теплый среднего до 37 ° С в ванну с горячей водой перед использованием. </li><li> Приготовьте 1х клеточную культуральную среду отдельно, как для нормальной клеточной культуре клеточной линии, представляющей интерес. </li><li> Приготовьте 1% агар благородный путем добавления 1 г благородный агар до 100 мл деионизированной воды. ПРИМЕЧАНИЕ: Благородный агар не растворится полностью при перемешивании в одиночку. </li><li> Подготовить0,6% благородный агар, добавив 0,6 г благородный агар в 100 мл деионизированной воды. Оба агар растворы могут быть сделаны в 100 мл стеклянных бутылках с закрывающимися крышками для длительного хранения. </li><li> Автоклав благородные агар смеси для стерилизации. Эти смеси могут быть сделаны заранее и хранили при 4 ° С, но следует снова нагревали в момент эксперимента, пока агар полностью не растворится. </li><li> Подготовьте нитросинем раствора хлорида тетразолия, сделав 1 мг / мл маточного раствора в 1х PBS (8 г NaCl, 0,2 г KCl, 1,44 г Na 2 HPO 4, и 0,24 г КН 2 РО 4 в H 2 O до конечного объема 1000 мл ). Это будет использоваться в конце эксперимента, чтобы окрасить колоний. </li></ol> 2. Покрытие из нижнего слоя агара <ol><li> Ослабить крышку на бутылке 1% благородного агар и микроволновой печью около 1-2 мин. При нагревании в микроволновой печи, следить за решение тесно, чтобы избежать при кипении. Продолжайте нагревание, при смешивании с перерывами,пока агар полностью не растворится и раствор не станет прозрачным. ПРИМЕЧАНИЕ: Используйте термостойкие перчатки для обработки колбу после нагревания. Несоблюдение этого требования может привести к ожогам или серьезным травмам. </li><li> Поместите расплавленный раствор агара и предварительно нагревают 2x культуральную среду в ледяной ведро с горячей водой (42 ° С). Кроме разместить 50 мл коническую трубку в держателе трубки в ведро со льдом с горячей водой. Трансфер ведро на капот культуре клеток для последующих шагов. </li><li> Для нижнем слое агара, понадобится 1,5 мл смеси агара и среды на лунку 6-луночного планшета. </li><li> Чтобы обеспечить достаточное количество смеси, готовят в общей сложности 12 мл для каждой 6-луночного планшета. </li><li> Начало добавлением 6 мл культуральной среды в 50 мл коническую пробирку, а затем 6 мл 1% раствора агара благородного. </li><li> Обратить конической трубе несколько раз перемешать. Работа в быстром темпе будет предотвратить преждевременное затвердевание мягкого агара. </li><li> Составьте примерно 5,5 мл смеси в 5 мл серологической пиPette. </li><li> Разрешить пузырьки воздуха, чтобы подняться до верхней части колонны пипетки перед нанесением 1,5 мл этой смеси в каждую лунку. Будьте осторожны, чтобы избежать осаждения пузырьков воздуха в лунки планшета. </li><li> Покрытие пластин агара и позволяют смеси затвердеть при комнатной температуре, в капот культуре клеток, в течение 30 мин. </li></ol> 3. Покрытие верхнего слоя агара, содержащие клетки <ol><li> После того, как нижний слой агара затвердеет, начать подготовку верхнего слоя. </li><li> Во-первых, урожай клеток путем обработки трипсином и разбавить их 1: 5 в культуральной среде (например, на 1 мл трипсина, добавить 4 мл среды) в 15 мл коническую пробирку. </li><li> Граф клеток и рассчитать количество клеток на лунку, необходимых для получения конечной суспензии клеток в это время. Это количество будет варьироваться в зависимости от типа клеток. Используйте 5000 клеток / лунку в качестве отправной точки и корректировать по мере необходимости. Для этого числа клеток, вы бы подготовить клеточной суспензии из 6667 клеток / мл (т.е. каждый вэйл получит 0,75 мл этой суспензии и 0,75 мл агара для общего объема 1,5 мл; концентрация клеток будет также разводили 1: 2 для конечного общего количества клеток в 5000). </li><li> Объем клеточной суспензии, необходимой на лунку 6-луночного планшета с вновь будет 1,5 мл. Подготовьте дополнительный клеточной суспензии на общую сумму 12 мл на 6-луночный планшет. </li><li> Melt 0,6% раствора агара в микроволновой печи, как указано выше, и поместите в ведре со льдом, содержащей горячую воду вместе с 50 мл коническую пробирку в держателе трубки и конечной суспензии клеток со стадии 3.3. </li><li> Трансфер ведро со льдом с топленым 0,6% агара в капот культуре клеток для последующих этапов. </li><li> Смешайте 0,6% агара и клеточной суспензии в соотношении 1: 1, готовит общий объем 12 мл на 6-луночный планшет. Потребуется 1,5 мл на лунку, но дополнительное должны быть сделаны, как описано выше. </li><li> Пипетка 6 мл клеточной суспензии в 50 мл коническую пробирку. </li><li> Затем добавляют 6 мл 0,6% агара к трубе. ПРИМЕЧАНИЕ: Температура этой смеси должна бытьхранится около 42 ° C, чтобы избежать преждевременного затвердевания и максимизировать выживание клеток. </li><li> Работа быстро, пипетка эту смесь 2-3 раза распространять клетки, затем составить 5,5 мл смеси в 5 мл серологические пипетки. </li><li> Разрешить пузырьки воздуха, чтобы подняться на вершину колонны пипетки перед нанесением 1,5 мл этой смеси в каждую лунку. Будьте осторожны, чтобы избежать осаждения пузырьков воздуха в лунки планшета. </li><li> Разрешить клеток / агар смесь затвердевать при комнатной температуре, в капот культуре клеток, в течение 30 мин перед помещением в 37 ° C увлажненном инкубаторе культуры клеток. </li><li> Время, необходимое для адекватного образования колоний варьирует для каждой клеточной линии, как правило, около 21 дней. </li><li> Слой ростовой среды следует поддерживать на верхнем слое агара, чтобы предотвратить высыхание. 100 мкл среды, дополненной два раза в неделю достаточно для этой цели. </li></ol> 4. окрашивания пластин и Подсчет колоний <ol><li> Пятно клетки, добавляя 200 _6; л тетразола раствора хлорида на лунку и инкубирования пластин в течение ночи при 37 ° С. </li><li> После того, как колонии окрашиваются, фотографировать скважин с использованием тепловизора и рассчитывать колонии, используя программное обеспечение для анализа изображений. </li></ol>

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The authors have nothing to disclose.

Экстракорпоральное подтверждения опухоли подавляющих функцию сигнальных белков трудно. Один из самых строгих анализов, доступных для расследования этого имущества является мягкий агар анализ образования колоний. Здесь мы проиллюстрировали мягкий агар анализа формирования кол...

Мягком агаре образование колоний анализ представляет собой метод широко используется для оценки клеточной трансформации в пробирке. Исторически сложилось так, другой тест, клоногенности, описывается шайба и др. В 1956 г. был использован для оценки способности клеток образовывать колонии 1. В этой технике, клетки были рассеяны на культуральную чашку и выращивают в присутствии &#39;&#39; фидерных клеток или кондиционированной среды, чтобы обеспечить необходимые ростовые факторы. Ограничение этого способа в том, что это только при условии, информацию в отношении образования колоний. Нормальные клетки не имеют возможности крепления-независимого роста, из-за того или иного вида апоптоза, называемой anoikis 2. Тем не менее, трансформированные клетки обладают способностью расти и делиться без привязки к подложке. Чтобы извлечь выгоду из этой концепции, исследователи разработали программную агар анализа формирования колонии. Мягком агаре анализ образование колоний с тех пор были изменены, в последние годы, для решения Specific потребности. Один из вариантов включает включение флуорометрического красителем, чтобы позволить для подсчета колоний высокой пропускной. Еще один вариант предполагает использование специализированного раствора агара, чтобы обеспечить извлечение жизнеспособных клеток после образования колоний, когда белок или ДНК пробы нужны. В традиционном мягком агаре колонии анализа пласта, клетки выращивают в слое мягком агаре, смешанного с клеточной культуральной среде, которая лежит на другом слое мягком агаре, также в смеси с клеточной культуральной среды, но содержащий более высокую концентрацию агара. Это предотвращает клетки от присоединения к культурального планшета, пока позволяет трансформированные клетки образуют видимые колонии. Смысл этого метода является то, что нормальные клетки зависит от клетки к внеклеточной матрицы контакте, чтобы иметь возможность расти и делиться. И наоборот, трансформированные клетки обладают способностью к росту и делению, независимо от окружающей их среды. Таким образом, клетки, способные образовывать колонии в опоре-независимым способом были Considered должны быть преобразованы и канцерогенными. Общая цель этого метода заключается в измерении эту возможность в клетках в полу-количественного и строгой манере. Сигнальный путь Wnt является критическим в эмбриогенезе и часто де-регулируется в онкогенеза 3-6. Есть несколько путей, связанные с Wnt сигнализации. Канонический путь включает Wnt сигнализацию и регулирование вниз по течению транскрипции генов через его влияние на транскрипционный коактиватора бета-катенина. Wnts также сигнализировать через несколько неканонические путей, например, плоской клеточной полярности путь, который регулирует элементы, участвующие в структуре цитоскелета 7, и путь Wnt-кальция, который регулирует высвобождение кальция из эндоплазматического ретикулума 8. Wnt лигандов проявляют свою активность посредством связывания Frizzled рецепторы. Хотя несколько Wnts, как было показано, чтобы быть активируется при раке легкого, Wnt7a было показано, что подавляется в не-Smalрак легких л клеток через метилирования промоторов 9. Wnt7a связывает Fzd9 и действует как подавитель опухоли через неканонической пути. Восстановление Wnt-7а и FZD-9 подавляет рост немелкоклеточного рака легкого ячеек 10. Эффекты Wnt7a / Fzd9 опосредованы активацией ERK-5, который, в свою очередь, активирует пероксисом γ-рецептора активатора пролиферации (PPAR, 11,12). Здесь мы показываем, что избыточная экспрессия Wnt7a и Fzd9 приводит к подавлению крепления независимый рост мышиной линии карциномы легкого клеток. Мышиные CMT167 клетки были получены из карциномы легких у C57BL мышей / 13 КЗоТ и были стабильно трансфицированные Wnt7a и Fzd9. Сверхэкспрессия Wnt7a и Fzd9 были подтверждены количественного-ПЦР (Q-PCR) и функциональности Wnt7a и Fzd9 гиперэкспрессией было подтверждено через потоку активации PPAR,.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Cancer Cell Line of Interest</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">10032302</td>
			<td>CMT-167 Cells</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Powdered RPMI 1640 Medium</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">31800-089</td>
			<td style="width:167px;">Used to prepare 2X cell culture medium.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Liquid RPMI 1650 Medium</td>
			<td style="width:71px;">Cellgro</td>
			<td style="width:119px;">10-040-CV</td>
			<td style="width:167px;">Referred to as 1X cell culture medium.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:71px;">HyClone</td>
			<td style="width:119px;">SH30910.03</td>
			<td style="width:167px;">Used to supplement cell culture medium.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:71px;">CellGro</td>
			<td style="width:119px;">30-002-Cl</td>
			<td style="width:167px;">Used in cell culture medium.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Difco Noble Agar</td>
			<td style="width:71px;">BD Biosciences</td>
			<td style="width:119px;">214230</td>
			<td style="width:167px;">Used to prepare 1.0% and 0.6% Agar.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sodium Bicarbonate</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">BP-328-1</td>
			<td style="width:167px;">Used in 2X cell culture medium.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Trypsin</td>
			<td style="width:71px;">Cellgro</td>
			<td style="width:119px;">25-050-Cl</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sterile Bottle-Top Filters</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">09-761-126</td>
			<td style="width:167px;">Used to sterile filter 2X medium.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Lipofectamine Reagent</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">18324-020</td>
			<td style="width:167px;">Used in PPAR-RE luciferase assay.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">6-well Plates Tissue-culture Treated</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">37 degree C/5% CO2 Incubator</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Chemi-Doc Imager</td>
			<td style="width:71px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Used to take pictures of colonies.&#160;&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Quantity One Software</td>
			<td style="width:71px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Used to count cell colonies.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">15mL Conical Tubes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">50mL Conical Tubes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">250mL Erlenmeyer Flasks</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Microwave</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">5mL Serological Pipettes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Pipette Aid</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Micropipette</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Hemacytometer w/ cover slip</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Pipette Tips</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Inverted Light Microscope</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Centrifuge</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Heat-Resistant Gloves</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Saran Wrap</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ice Bucket</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

the soft agar colony formation assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

Выражение Wnt7a и Fzd9 в CMT167 клеток является эффективным в подавлении опухолевого как свидетельствует наша мягкой агар анализа формирования колонии. Предварительно, мы использовали Q-PCR, чтобы показать, что Wnt7a и Fzd9 мРНК выражены в низких уровнях в CMT167 клеток. CMT167 клетки показали низкий уров?...

Watch this Scientific Journal Video about The Soft Agar Colony Formation Assay at JoVE.com

The Soft Agar Colony Formation Assay

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

Формирование Анализ Soft Агар колонии

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

Research

JoVE Journal

Biology

110.6K Views.  University of Illinois at Chicago. The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

Video: The Soft Agar Colony Formation Assay

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

Анализ для адгезии и агар Вторжение в CEREVISIAE С.

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions between lymphocytes and antigen presenting cells, we use Ni2+-chelating lipids to anchor recombinant cell adhesion and MHC proteins to the upper leaflet of a bilayer with poly-histidine tags. Planar bilayers are generated by preparing lipid, treating the glass surfaces where the bilayer will form, and then forming the bilayer in a specialized chamber containing a flow-cell where the lymphocytes will be added.  Then, bilayers are charged with Ni and his-tagged recombinant proteins are added.  Finally,  lymphocytes are injected into the flow cell and TIRF microscopy can be used to image synapse formation and the mechanisms that control T cell locomotion, sites of receptor sorting,  and sites of receptor degradation.

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. Here, we show methods for anchoring cell adhesion proteins known to modulate synapse formation to the upper leaflet of the lipid bilyer and visualize synapse formation using TIRF microscopy.

Поддерживаемые Planar Бислои для формирования Исследование иммунологических синапсов и Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions ...

Isolating Stem Cells from Soft Musculoskeletal Tissues

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of excitement for treating injured and diseased tissues due to their impressive capabilities to undergo multi-lineage cell differentiation and their self-renewal ability. Most importantly, these qualities have made them advantageous for use in autologous cell transplantation therapies. The current protocol will introduce the readers to the modified preplate technique where soft tissues of the musculoskeletal system, e.g. tendon and muscle, are 1st enzymatically dissociated and then placed in collagen coated flasks with medium. The supernatant, which is composed of medium and the remaining floating cells, is serially transferred daily to new flasks. The stem cells are the slowest to adhere to the flasks which is usually takes 5-7 days (serial transfers or preplates) . By using this technique, adult stem cells present in these tissues can be easily harvested through fairly non-invasive procedures.

Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask.

Изоляция стволовых клеток из мягких тканей опорно-двигательного

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

RhoC GTPase Activation Assay

RhoC GTPase has 91% homology to RhoA GTPase. Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed. However, RhoC GTPase is expressed in metastatic cancer cells at relatively low levels. Therefore, few RhoC-specific reagents have been developed. We have adapted a Rho activation assay to detect RhoC GTPase. This technique utilizes a GST-Rho binding domain fusion protein to pull out active RhoC GTPase. In addition, we can harvest total protein at the beginning of the assay to determine levels of total (GTP and GDP bound) RhoC GTPase. This allows for the determination of active versus total RhoC GTPase in the cell. Several commercial versions of this procedure have been developed however, the commercial kits are optimized for RhoA GTPase and typically do not work well for RhoC GTPase. Parts of the assay have been modified as well as development of a RhoC-specific antibody.

This protocol utilizes a pull down assay to determine the levels of active RhoC GTPase within cells.

RhoC GTPase Активация Пробирной

RhoC GTPase has 91% homology to RhoA GTPase.  Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed.  ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

Дрожжи колонии Вложение Метод

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Клоногенных Пробирной: прилипшие клетки

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Матригель основе труб Формирование Пробирной для оценки Vasculogenic активность опухоли

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

Количественный анализ на инсулиноподобный выражения колониеобразующих прародителей

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses 1. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities 2. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media 3. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis 4, and Gram-negative bacteria, such as Vibrio cholerae 5, Vibrio parahaemolyticus 6, Pseudomonas aeruginosa 7, and Vibrio fischeri 8.
The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes 8-10. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect 9,11, while strains exhibiting increased biofilm phenotypes are enhanced for colonization 8,12. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization.
In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

Полу-количественный подход к оценке биопленки использование морщинистой развития колонии

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Эндотелия Формирование Анализ сотового трубка для<em&gt; In Vitro</em&gt; Изучение ангиогенеза

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...