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

1.准备材料和试剂<ol><li>标记的组织培养的各孔处理的6孔板适当地对每个细胞系或状况进行调查。 </li><li>通过将1g粉末介质和0.2g碳酸氢钠在去离子水至50ml的终体积制备2倍的细胞培养基。 </li><li>通过0.2微米的过滤器进行消毒过此介质。 </li><li>添加所需的感兴趣的细胞系的正常培养的额外组件。例如，生长在RPMI 1640培养基中补充有10％FBS和1％青霉素/链霉素溶液的CMT 167细胞系。温暖的培养基至37℃，在热水浴中在使用之前。 </li><li>另外准备1个细胞培养液中，你会感兴趣的细胞系正常的细胞培养。 </li><li>制备1％的贵金属琼脂中加入1克贵金属琼脂至100毫升的去离子水中。 注：带搅拌贵族琼脂不会完全溶解孤单。 </li><li>准备0.6％贵金属琼脂，加入将0.6克贵金属琼脂至100毫升的去离子水。既琼脂溶液可以在100毫升玻璃瓶闭自如的盖用于长期存储制成。 </li><li>高压灭菌高贵的琼脂混合消毒。这些混合物可以制成事先并保存于4℃，但应在实验的时间再次加热至琼脂完全溶解。 </li><li>制备氮蓝四唑氯化物溶液通过制备1mg / ml的储备液中的1×PBS（8克NaCl，0.2克氯化钾，1.44克的Na 2 HPO 4，及0.24g的KH 2 PO 4的H 2 O操作1000 ml终体积）。这将用于在实验结束时进行染色的菌落。 </li></ol> 2.电镀琼脂底层的<ol><li>松开瓶的1％贵金属琼脂和微波盖约1-2分钟。而加热的微波炉，监控解决方案紧密，以避免沸腾了。继续加热，同时混合间歇直到琼脂完全溶解，溶液澄清。 注：使用耐热手套加热后处理烧瓶中。如果不这样做可能会造成烧伤或严重伤害。 </li><li>放置熔化的琼脂溶液和预热的2×培养基中冰桶充满热自来水（42℃）。也放置在50ml锥形管中，在冰桶用热水的管座。转斗细胞培养罩的后续步骤。 </li><li>对于琼脂的底层，则需要1.5毫升琼脂的混合和中每一个6孔板的孔的。 </li><li>以确保混合物的足够量，制备总共12毫升每6孔板中。 </li><li>开始通过加入6毫升培养介质向50ml锥形管中，然后6毫升1％贵金属琼脂溶液中。 </li><li>倒置的锥形管几次以混合。工作在轻快的步伐将防止软琼脂过早硬化。 </li><li>制定约5.5毫升混合物到5ml的血清学圆周率佩特。 </li><li>使气泡上升到移液管塔的顶部以及沉积1.5ml该混合物到每个前。请小心，以避免任何气泡沉积到板孔。 </li><li>覆盖板并允许琼脂混合物固化在室温下，在细胞培养罩，30分钟。 </li></ol> 3.电镀琼脂含细胞的上层<ol><li>一旦琼脂的下层已经凝固，开始制备上层的。 </li><li>首先，收获的细胞用胰酶消化并稀释它们1:5的培养基（ 例如，1 ml胰蛋白酶的，加入4毫升培养基）入15ml锥形管中。 </li><li>计数细胞，并计算出每孔在这个时候制备的最终细胞悬浮液所需要的细胞数量。这个数目将取决于细胞类型而有所不同。使用5000细胞/孔为起点，并根据需要进行调整。对于这个手机号码，您将编写的6667个细胞/ ml的细胞悬液（ 即每个WEL升将接收0.75毫升此悬浮液加入0.75ml琼脂为1.5毫升总体积;细胞的浓度也将1:2稀释为5000的最终总细胞计数）。 </li><li>每一个6孔板的所需要的细胞悬浮液的体积将再次为1.5毫升制备额外的细胞悬浮液共计每6孔板12毫升</li><li>熔体在微波如上0.6％琼脂溶液中，并放入冰桶含热水连同50ml锥形管中的管保持器和来自步骤3.3的最终细胞悬浮液。 </li><li>转让融化的0.6％琼脂细胞培养罩的后续步骤的冰桶。 </li><li>混合0.6％的琼脂和细胞悬浮液以1：1的比例，制备12毫升每6孔板的总体积。 1.5毫升将被要求每孔加，但应作出以上。 </li><li>吸管6毫升细胞悬浮到50ml锥形管中。 </li><li>然后，添加6毫升0.6％琼脂的在管。 注：该混合物的温度必须保持在42℃，以避免过早硬化，并最大限度细胞的存活。 </li><li>工作迅速，吸这种混合物2-3倍分发细胞，然后制定5.5毫升混合成5毫升血清吸管。 </li><li>允许任何气泡上升到移液管塔的顶部以及沉积1.5ml该混合物到每个前。请小心，以避免任何气泡沉积到板孔。 </li><li>允许细胞/琼脂混合物放置到37℃的湿润的细胞培养箱中之前，在室温下固化，在细胞培养罩，30分钟。 </li><li>所需的足够的集落形成的时间而变化的每个细胞系，通常约21天。 </li><li>应维持在琼脂上以防止干燥的上层培养基中的层。将100μl加入每周两次培养基足以用于此目的。 </li></ol> 4.染色板和菌落计数<ol><li>染色的细胞中加入200 _6;微升每孔并过夜孵育板在37℃，氮蓝四唑氯化物溶液。 </li><li>一旦菌落染色，采取使用成像井的照片，并使用计数图像分析软件的殖民地。 </li></ol>

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The authors have nothing to disclose.

中的信号蛋白的肿瘤抑制功能的体外确认是困难的。一个可用来调查该属性的最严格的测定法是软琼脂集落形成测定。这里，我们已经说明了利用鼠肺癌细胞稳定过表达WNT7A和Fzd9相比其亲CMT167细胞系的软琼脂集落形成测定。 有考虑有关软琼脂法的几个要点。在此测定法中最关键的步骤是电镀的细胞。细胞计数必须是准确的，并将琼脂溶液不能是太热了。如果没?...

软琼脂集落形成测定是广泛用于评估体外细胞转化的技术。在历史上，另一个试验中，集落形成试验，通过冰球等人在1956年描述。用于评价细胞形成集落1的能力。在该技术中，将细胞分散到培养板中，在“进料器”细胞或条件培养基的存在下，以提供必要的生长因子生长。这种技术的局限性是，它仅对于集落形成提供信息。正常细胞是从锚定非依赖性生长抑制，由于特定类型的凋亡性死亡，称为失巢2。然而，转化细胞有能力生长和分裂，而不结合到基片上。利用这一概念，研究人员开发出了软琼脂克隆形成实验。软琼脂集落形成测定法进行了修改，在最近几年，为解决藻ecific需求。一个变化涉及到荧光染料的掺入，允许高通量菌落计数。另一种变型涉及使用专门的琼脂溶液中，以允许集落形成后检索的活细胞时，需要的蛋白质或DNA样本。 在传统的软琼脂集落形成测定法，细胞生长在一层软琼脂与搁置在软琼脂中，还混有细胞培养基的另外一个层细胞培养基混合的，但含有琼脂的浓度较高。这可以防止细胞粘附到培养板上，还允许转化的细胞形成可见的菌落。该技术背后的基本原理是，正常细胞依赖于细胞外基质接触，以便能够生长和分裂。相反，转化细胞具有生长和分化而不论其周围环境的能力。因此，细胞能够形成菌落在锚地无关的方式为Considered待转化和致癌性。该方法的总体目标是测量在一个半定量的和严格的方式在细胞中这种能力。 Wnt信号通路是在胚胎发育的关键和经常去调节肿瘤发生3-6。有与Wnt信号相关联的多种途径。规范的途径，通过对转录共激活因子的β-catenin的影响涉及Wnt信号和下游基因的转录调控。 Wnts还通过几个非经典途径的信号，例如，在平面细胞极性通路，它调节参与细胞骨架结构7的元素，并且所述的Wnt-钙通路，调节钙的释放从内质网8。的Wnt配体通过结合型卷曲受体发挥其活性。虽然几个Wnts已被证明在肺癌中上调，WNT7A已显示被下调非SMaL公司通过启动子甲基化9升细胞肺癌。 WNT7A结合Fzd9并作为通过非经典途径中的肿瘤抑制基因。的Wnt-7a和FZD-9的恢复抑制非小细胞肺癌细胞10的生长。 WNT7A / Fzd9的作用是通过ERK-5，这反过来，激活过氧化物酶体增殖物激活受体γ（PPARγ）11,12的激活介导的。在这里，我们表明WNT7A和Fzd9的过度表达导致的小鼠肺癌细胞株的锚定非依赖性生长的抑制。鼠CMT167细胞来自于C57BL / lcrf小鼠13个肺腺癌和稳定转染WNT7A和Fzd9。 WNT7A和Fzd9的过表达通过定量PCR（Q-PCR）和WNT7A和Fzd9过表达的功能性被证实是通过PPARγ的下游激活确认。

<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.

如由我们的软琼脂集落形成测定WNT7A和Fzd9在CMT167细胞中的表达是有效的肿瘤抑制。预先，我们用Q-PCR表明，WNT7A和Fzd9 mRNA的表达在低的水平在CMT167细胞。 CMT167细胞表现出内源性WNT7A和Fzd9水平低时相比，MLE-12细胞中，SV40转化的小鼠肺上皮细胞系（ 图1）。然后，我们转CMT167细胞2的逆转录病毒表达载体表达WNT7A（LNCX-WNT7A）和Fzd9（LPCX-Fzd9）的人构建体来创建表示既WNT7A和Fzd9（CMT LL WNT7A / Fzd9...

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.

软琼脂集落形成分析

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

110.8K 次观看.  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.

视频: 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.

化验粘附和酿酒酵母琼脂入侵

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

科研

生物学

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.

形成免疫突触和Kinapse研究的支持平面双层膜

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.

从软肌肉骨骼组织中分离干细胞

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

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; agar-block). It is well-accepted that soil-block microcosms yield higher decay rates, fewer moisture issues, lower variability among studies, and higher thresholds of preservative toxicity. Soil-block testing is thus the more utilized technique and has been standardized by American Society for Testing and Materials (ASTM) (method D 1413-07). The soil-block design has drawbacks, however, using locally-variable soil sources and in limiting the control of nutrients external (exogenous) to the decaying tissues. These drawbacks have emerged as a problem in applying this method to other, increasingly popular research aims. These modern aims include degrading lignocellulosics for bioenergy research, testing bioremediation of co-metabolized toxics, evaluating oxidative mechanisms, and tracking translocated elements along hyphal networks. Soil-blocks do not lend enough control in these applications. A refined agar-block approach is necessary.
Here, we use the brown rot wood-degrading fungus Serpula lacrymans to degrade wood in agar-block microcosms, using deep Petri dishes with low-calcium agar. We test the role of exogenous gypsum on decay in a time-series, to demonstrate the utility and expected variability. Blocks from a single board rip (longitudinal cut) are conditioned, weighed, autoclaved, and introduced aseptically atop plastic mesh. Fungal inoculations are at each block face, with exogenous gypsum added at interfaces. Harvests are aseptic until the final destructive harvest. These microcosms are designed to avoid block contact with agar or Petri dish walls. Condensation is minimized during plate pours and during incubation. Finally, inoculum/gypsum/wood spacing is minimized but without allowing contact. These less technical aspects of agar-block design are also the most common causes of failure and the key source of variability among studies. Video publication is therefore useful in this case, and we demonstrate low-variability, high-quality results.

This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.

为控制植物组织分解好氧菌的琼脂块缩影

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

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.

酵母殖民地嵌入方法

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.

克隆形成含量：贴壁细胞

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.

基于一个基质胶管形成实验，以评估对肿瘤细胞的血管生成活性

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.

一个定量测定胰岛素表达细胞集落形成先觉

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.

一个半定量的方法来评估生物膜形成皱殖民地的发展

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.

内皮细胞小管形成实验的<em&gt;在体外</em&gt;研究血管生成

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