Name: the soft agar colony formation assay
Uploaded: 2014-10-27T15:00:00.000+00:00
Duration: 8 min 1 s
Description: Watch this Scientific Journal Video about The Soft Agar Colony Formation Assay at JoVE.com

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

材料および試薬の調製<ol><li>組織培養の各ウェルは、調査されて各細胞株または状態に対して適切に6ウェルプレートを処理したラベル。 </li><li>粉末培地1gを、50mlの最終体積まで脱イオン水に炭酸水素ナトリウム0.2gを溶解することにより2倍の細胞培養培地を準備する。 </li><li>滅菌するために0.2μmのフィルターを介してこのメ​​ディアを渡します。 </li><li>関心のある細胞株の通常の培養に必要な追加コンポーネントを追加します。たとえば、10％FBSおよび1％ペニシリン/ストレプトマイシン溶液を補充したRPMI 1640培地中でCMT 167細胞株を成長させる。湯浴中で37℃に温培地使用前に。 </li><li>あなたが興味のある細胞株の正常な細胞培養の場合と同じように別々の1x細胞培養培地を準備します。 </li><li>脱イオン水100mlに希寒天1gを添加することにより、1％希寒天を準備する。 注：ノーブル寒天単独で攪拌しながら完全に溶解しない。 </li><li>準備する脱イオン水100mlに希寒天0.6gを添加することにより寒天希0.6％。両方の寒天溶液は、長期保存のための閉鎖可能な蓋付きの100mlのガラス瓶中で行うことができる。 </li><li>殺菌するために高貴な寒天の混合物をオートクレーブ。これらの混合物は、事前に作製し、4℃で保存したが、寒天が完全に溶解するまで、実験の際に再加熱されるべきであることができる。 </li><li>千ミリリットルの最終容量に8gのNaCl、0.2gのKCl、1.44グラムHPO 4 2のNa、0.24グラムのKH H 2 Oで2 PO 4（1×PBS中1mg / mlのストック溶液を作製することにより、ニトロブルーテトラゾリウムクロリド溶液を調製）。これは、コロニーを染色するために、実験の終了時に使用される。 </li></ol>寒天の底層の2めっき<ol><li>約1〜2分間、1％高貴寒天およびマイクロ波のボトルのキャップを緩めます。電子レンジで加熱しながら、オーバー沸騰を避けるために密接にソリューションを監視します。断続的に混合しながら、加熱を継続し、寒天を完全に溶解されるまで、溶液は透明である。 注：加熱後にフラスコを処理するために、耐熱手袋を使用してください。そうしないと、火傷や重傷を引き起こす可能性があります。 </li><li>熱い水道水（42℃）で満たされたアイスバケット内で溶融した寒天液と予め温め2xの培養液を置きます。また、お湯でアイスバケット内チューブホルダーに50ミリリットルコニカルチューブを配置します。後続のステップのための細胞培養フードにバケツを転送します。 </li><li>寒天の最下層では、6ウェルプレートのウェルあたり寒天培地の混合液1.5mlを必要とする。 </li><li>混合物の十分な量を確保するために、各6ウェルプレート、12 mLの合計を準備する。 </li><li> 50mlのコニカルチューブに培地を6mlを添加することによって開始し、次いで1％の希寒天溶液を6ml。 </li><li>混合するコニカルチューブを数回転倒。活発なペースで働くこと軟寒天の早すぎる硬化を防ぐことができます。 </li><li> 5ミリリットルの血清学的パイに、混合物の約5.5ミリリットルを策定pette。 </li><li>気泡が各ウェルにこの混合物に、1.5mlの堆積前に、ピペットカラムの上部に上昇することを可能にする。プレートウェルに気泡の付着を避けるために注意してください。 </li><li>プレートをカバーし、寒天混合物を30分間、細胞培養フード内で、室温で固化することを可能にする。 </li></ol> 3.細胞を含む寒天の上層めっき<ol><li>寒天下層が凝固した後、上層の準備を開始する。 </li><li> （トリプシンの1ミリリットル用など培地4mlを加える）培地中で5 15ミリリットルコニカルチューブに：まず、トリプシン処理によって収穫細胞とは、彼らに1を希釈。 </li><li>細胞をカウントし、この時点で最終的な細胞懸濁液を調製し、ウェル当たりに必要な細胞の数を計算する。この数は、細胞型に依存して変化する。 5000細胞/ウェルとしての出発点を使用し、必要に応じて調整します。この細胞数のために、 すなわち 、各welの（6667細胞/ mlの細胞懸濁液を調製うlはこの懸濁液の0.75ミリリットルと1.5ミリリットルの全容積のための寒天の0.75ミリリットルを受け取ります。 5000最後の総細胞数）が2：細胞の濃度はまた、1に希釈される。 </li><li> 6ウェルプレートのウェルあたりに必要な細胞懸濁液の量が再び1.5ミリリットルであろう。 6ウェルプレート当たり12ミリリットル合計追加の細胞懸濁液を準備します。 </li><li>上記のように電子レンジで0.6％寒天溶液を融解し、ステップ3.3からのチューブホルダーおよび最終細胞懸濁液に50mlの円錐チューブに沿ってお湯を含むアイスバケットに入れる。 </li><li>後続のステップのための細胞培養フードに溶融した0.6％の寒天をアイスバケットを転送します。 </li><li> 6ウェルプレートあたり12 mLの総体積を調製1：1の比で0.6％寒天および細胞懸濁液を混合する。 1.5ミリリットルウェルあたり必要とされますが、余分な上記のようになされるべきである。 </li><li>ピペット50mlコニカルチューブに細胞懸濁液を6ml。 </li><li>その後、チューブに0.6％寒天の6ミリリットルを追加します。 注：この混合物の温度でなければならない時期尚早の硬化を回避し、細胞の生存を最大化するために周りに42°Cに保った。 </li><li>ピペットの細胞を分配するためにこの混合物を2〜3倍、迅速に作業し、その後5ミリリットルの血清学的ピペットに、混合物の5.5ミリリットルを策定。 </li><li>気泡が各ウェルにこの混合物を1.5mlの堆積前に、ピペットカラムの上部に上昇することを可能にする。プレートウェルに気泡の付着を避けるために注意してください。 </li><li>セル/寒天混合物を37℃で加湿細胞培養インキュベーター中に配置する前に30分間、細胞培養フード内で、室温で固化することを可能にする。 </li><li>適切なコロニー形成に要する時間は、約21日、典型的には、各細胞株ごとに異なる​​。 </li><li>成長培地の層は乾燥を防ぐために、寒天の上層の上に維持されるべきである。週二回添加した培地100μlのは、この目的のために十分である。 </li></ol> 4.プレートを染色し、コロニー数を計測<ol><li> 200を追加することによって染色細胞_6;ウェル当たりニトロブルーテトラゾリウムクロリド溶液リットル、37℃で一晩プレートをインキュベートする。 </li><li>コロニーを染色すると、撮像装置を使用して、ウェルを撮影し、画像解析ソフトウェアを用いてコロニーを数え。 </li></ol>

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The authors have nothing to disclose.

シグナル伝達タンパク質の腫瘍抑制機能のインビトロでの確認は困難である。このプロパティを調査するために利用できる最も厳格なアッセイの1つは、軟寒天コロニー形成アッセイである。ここでは、安定的にその親CMT167細胞株と比較して、Wnt7aおよびFZD9を過剰発現するマウス肺癌細胞株を用いて、軟寒天コロニー形成アッセイを例示している。 軟?...

軟寒天コロニー形成アッセイは、広く、インビトロで細胞形質転換を評価するために使用される技術である。歴史的に、別のアッセイは、パックらによって記載され 1956年にクローン原性アッセイは、コロニー1を形成する細胞の能力を評価するために使用した。この技術では、細胞を培養プレート上に分散させ、必要な成長因子を提供するための「フィーダー」細胞または馴化培地の存在下で増殖させた。この技術の制限は、それが唯一のコロニー形成に関する情報を提供したことであった。正常細胞は、アノイキス2と呼ばれるアポトーシス死の特定のタイプに、足場非依存性増殖を防止することができる。しかし、形質転換細胞は、基質に結合することなく成長し、分裂する能力を有する。この概念を活用するために、研究者らは軟寒天コロニー形成アッセイを開発しました。軟寒天コロニー形成アッセイは、以降のspに対応するために、より近年では、改変されているecificニーズ。一変形形態では、ハイスループットコロニー計数を可能にするために、蛍光色素の取り込みを含む。別の変形例は、タンパク質またはDNAのサンプルが必要な場合にコロニーを形成した後の生存細胞の回収を可能にするために特殊な寒天溶液の使用を含む。 従来の軟寒天コロニー形成アッセイにおいて、細胞は、軟寒天の別の層上に載っている細胞培養培地を含む軟寒天混合物の層で増殖される細胞培養培地と混合したが、寒天、より高い濃度で含有する。これは、培養プレートに付着する細胞を防ぎ、まだ目に見えるコロニーを形成する細胞を形質転換することを可能にする。この手法の背後にある理論的根拠は、正常細胞が成長し、分裂することができるように、細胞外マトリックスへの細胞接触に依存することである。逆に、形質転換された細胞は成長し、関係なく周囲の環境の分裂能を有している。したがって、足場非依存性の方法でコロニーを形成することができる細胞は、cた形質転換されるonsideredと発がん性。この方法の全体的な目標は、半定量的かつ厳格な方法で、細胞内でこの機能を測定することである。 Wntシグナル伝達経路は、胚発生に重要な、しばしば腫瘍形成3-6で脱調節されている。 Wntシグナル伝達に関連する複数の経路がある。標準経路は、転写コアクチベータカテニンに対するその効果を通してWntシグナル伝達および下流の遺伝子転写の調節を必要とする。 Wntはまた、例えば、いくつかの非標準的な経路を介して細胞骨格構造7、および小胞体8からのカルシウムの放出を調節するのWnt-カルシウム経路に関与する要素を調節する平面細胞極性経路を、信号。 Wntシグナルリガンドが結合フリッツルド受容体を介してその活性を発揮する。いくつかのWntが肺癌において上方制御されることが示されているが、Wnt7a、非Smalの中でダウンレギュレートされることが示されているプロモーターのメチル化9を介してLの細胞肺癌。 Wnt7aはFZD9を結合し、非標準的経路を介して腫瘍抑制因子として機能します。のWnt-7aおよびFZD-9の回復は、非小細胞肺癌細胞10の成長を阻害する。 Wnt7a / FZD9の効果は、ERK-5、今度は、ペルオキシソーム増殖因子活性化受容体γ（PPARγ）11,12を作動させるの活性化を介して媒介される。ここでは、Wnt7aおよびFZD9の過剰発現は、マウス肺癌細胞株の足場非依存性増殖の抑制をもたらすことを示している。マウスCMT167細胞はC57BL / lcrfマウス13で肺癌に由来しかつ安定Wnt7AおよびFZD9トランスフェクトした。 Wnt7AとFZD9の過剰発現は、定量的PCR（Q-PCR）によって確認したとWnt7AとFZD9過剰発現の機能は、PPARγの下流の活性化を介して確認された。

<table><tbody><tr><td>Cancer Cell Line of Interest</td><td>Sigma-Aldrich</td><td>10032302</td><td>CMT-167 Cells</td></tr><tr><td>Powdered RPMI 1640 Medium</td><td>Gibco</td><td>31800-089</td><td>Used to prepare 2x cell culture medium.</td></tr><tr><td>Liquid RPMI 1650 Medium</td><td>Cellgro</td><td>10-040-CV</td><td>Referred to as 1x cell culture medium.</td></tr><tr><td>Fetal Bovine Serum</td><td>HyClone</td><td>SH30910.03</td><td>Used to supplement cell culture medium.</td></tr><tr><td>Penicillin/Streptomycin</td><td>CellGro</td><td>30-002-Cl</td><td>Used in cell culture medium.</td></tr><tr><td>Difco Noble Agar</td><td>BD Biosciences</td><td>214230</td><td>Used to prepare 1.0% and 0.6% agar.</td></tr><tr><td>Sodium Bicarbonate</td><td>Fisher</td><td>BP-328-1</td><td>Used in 2x cell culture medium.</td></tr><tr><td>Trypsin</td><td>Cellgro</td><td>25-050-Cl</td><td></td></tr><tr><td>Sterile Bottle-Top Filters</td><td>Fisher</td><td>09-761-126</td><td>Used to sterile filter 2x medium.</td></tr><tr><td>Lipofectamine Reagent</td><td>Invitrogen</td><td>18324-020</td><td>Used in PPAR-RE luciferase assay.</td></tr><tr><td>6-well Plates Tissue-culture Treated</td><td></td><td></td><td></td></tr><tr><td>37 &amp;#176;C/5% CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td></td><td></td><td></td></tr><tr><td>Chemi-Doc Imager</td><td>Bio-Rad</td><td></td><td>Used to take pictures of colonies.</td></tr><tr><td>Quantity One Software</td><td>Bio-Rad</td><td></td><td>Used to count cell colonies.</td></tr><tr><td>15 ml Conical Tubes</td><td></td><td></td><td></td></tr><tr><td>50 ml Conical Tubes</td><td></td><td></td><td></td></tr><tr><td>250 ml Erlenmeyer Flasks</td><td></td><td></td><td></td></tr><tr><td>Microwave</td><td></td><td></td><td></td></tr><tr><td>5 ml Serological Pipettes</td><td></td><td></td><td></td></tr><tr><td>Pipette Aid</td><td></td><td></td><td></td></tr><tr><td>Micropipette</td><td></td><td></td><td></td></tr><tr><td>Hemacytometer w/ cover slip</td><td></td><td></td><td></td></tr><tr><td>Pipette Tips</td><td></td><td></td><td></td></tr><tr><td>Inverted Light Microscope</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td></td><td></td><td></td></tr><tr><td>Heat-Resistant Gloves</td><td></td><td></td><td></td></tr><tr><td>Saran Wrap</td><td></td><td></td><td></td></tr><tr><td>Ice Bucket</td><td></td><td></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.

我々の軟寒天コロニー形成アッセイによって示されるようにCMT167細胞におけるWnt7AおよびFZD9の発現は、腫瘍抑制に有効である。予め、我々はWnt7AおよびFZD9 mRNAがCMT167細胞において低レベルで発現されることを示すためにQ-PCRを用いた。 MLE-12細胞は、SV40形質転換マウス肺上皮細胞株（ 図1）と比較した場合CMT167細胞は、内因性Wnt7AおよびFZD9の低いレベルを示した。私たちは、その後...

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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.

軟寒天コロニー形成アッセイ

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

111.3K ビュー.  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.

ビデオ: 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.

S. cerevisiaeの接着と寒天の侵略のためのアッセイ

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

生物学

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

乳癌細胞における腫瘍形成の阻害剤を同定するための軟寒天コロニー形成アッセイの活用

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

医学

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

ソフト寒天オーバーレイ技術の使用は、細菌産生阻害する化合物をスクリーニングするために

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

免疫・感染学

Aseptic Laboratory Techniques: Plating Methods

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) as well as Material Safety Data Sheets (MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection (ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to: 
 
&#9679; Perform plating procedures without contaminating media. 
&#9679; Isolate single bacterial colonies by the streak-plating method. 
&#9679; Use pour-plating and spread-plating methods to determine the concentration of bacteria. 
&#9679; Perform soft agar overlays when working with phage. 
&#9679; Transfer bacterial cells from one plate to another using the replica-plating procedure. 
&#9679; Given an experimental task, select the appropriate plating method.

When working with media and reagents used to culture microorganisms, aseptic technique must be practiced to ensure contamination is minimized. A variety of plating methods are routinely used to isolate, propagate, or enumerate bacteria and phage, all of which incorporate procedures that maintain the sterility of experimental materials.

無菌実験技術：めっき法

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies ...

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.

インスリン発現コロニーを形成する前駆細胞の定量アッセイ

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.

しわコロニーの開発を使用したバイオフィルム形成を評価するための半定量的アプローチ

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

Keratinocyte Colony Formation Assay: An In Vitro Assay to Assess the Ability of Keratinocytes to Grow into Colonies

Source: Morris, R. J. et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/58701"> Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture</a>. J. Vis. Exp. (2019)
In this video, we perform a clonogenic assay of keratinocytes harvested from adult mice to determine the ability of individual cells to proliferate and form colonies. This keratinocyte harvesting technique is useful for studying other downstream aspects of cutaneous chemical carcinogenesis and tumor promotion.

Source: Morris, R. J. et al.  Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture. J. Vis. Exp. (2019)
In this video, we ...

JoVE Encyclopedia of Experiments

がん研究

皮膚がん

アッセイと技術

Soft Agar Colony Formation Assay: A Method to Test Effects of Novel Compounds on Cancer Cell Proliferation

Source: Horibata A. et. al., <a href="https://jove.unicartagenaproxy.elogim.com/v/52727/utilization-soft-agar-colony-formation-assay-to-identify-inhibitors"> Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells.</a> J. Vis. Exp. (2015).
This video describes a soft agar colony formation assay to test the effects of a PADI enzyme inhibitor and BB-Cl-amidine on breast cancer tumorigenicity in vitro. This assay also enables the quantitative assessment of cell transformation potential within the context of genetic perturbations.

軟寒天コロニー形成アッセイ:癌細胞増殖に対する新規化合物の影響をテストする方法

Source: Horibata A. et. al.,  Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells. J. Vis. ...

乳がん

試験管研究

Colony Formation Assay: Assessing the Efficacy of Anticancer Agents on Colony-Forming Lung Cancer Cells

Source: Lee, J. Y.et al, <a href="https://jove.unicartagenaproxy.elogim.com/v/57490/preclinical-assessment-bioactivity-anticancer-coumarin-ot48-spheroids">Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts.</a>&#160;J. Vis. Exp.(2018).
This video describes the colony formation assay, which helps determine lung cancer cells&#39; ability to proliferate indefinitely and form individual colonies. We can analyze the effect of an anticancer agent on the colony-forming cells by analyzing the changes in the number and/or size of the resulting colonies.

コロニー形成アッセイ:コロニー形成肺がん細胞に対する抗がん剤の有効性の評価

Source: Lee, J. Y.et al, Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish ...

肺がん

A Charcoal Agar Resazurin Assay to Evaluate Test Compound Activity against Mycobacteria

Inoculate polypropylene round-bottom or conical centrifuge tubes containing 4 or 20 milliliters respectively of Middlebrook 7H9-ADN, or 7H9-OADC with M. smegmatis at an OD580 of 0.01 to 0.1. Incubate the M. smegmatis cultures at 37 degrees Celsius and 20% O2 with shaking, expanding the cultures to mid-log phase or an approximate OD580 of 0.5.
To set up a minimal inhibitory concentration style experiment under replicating and non-replicating conditions, distribute 200 microliters of cells in 7H9-ADN at an OD580 of 0.01 into all the wells of a clear-bottomed tissue culture-treated 96-well plate.
For the non-replicating assay, use PBS containing 0.02% tyloxapol to wash the cells twice and use non-replicating medium to resuspend the cells. Use non-replicating medium to dilute the cells to an OD580 of 0.1, and add sodium nitrite to a final concentration of 0.5 to 5 millimolar from a freshly prepared 1 molar stock.
Distribute 200 microliters of cells at an OD580 of 0.1 into all wells of a clear-bottomed tissue culture-treated 96-well plate. After preparing test agents according to the text protocol, add 2 microliters of test agent #1 into rows A through E, and 2 microliters of test agent #2 into rows E through H, and mix thoroughly.
For replicating assays, incubate M. smegmatis microplates at 37 degrees Celsius, 20% O2, and 5% CO2 for 1 to 48 hours. At the time points in which the CARA will be used as a readout, use a P200 multichannel pipette set at 50 to 75 microliters to carefully resuspend the well contents of the MIC90-style assay plate by pipetting up and down 5 to 10 times. Then, use the pipette tips to gently swirl the contents of the wells in a circular motion.
Transfer 10 microliters of the undiluted assay well contents to the corresponding wells of the CARA microplate, avoiding splashing during transfers, and ensuring that the 10-microliter aliquot is spotted into the middle of the CARA microplate wells. With plate tape, bind stacks of CARA microplates, and then, place them into a resealable plastic bag. Incubate the M. smegmatis CARA microplates at 37 degrees Celsius with 20% O2 for one to two days for replicating plates and for two to three days for non-replicating plates.
When a film of bacterial growth, or larger macroscopic colonies are visible in the negative control wells, use a single set of 12 P200 tips with a multichannel pipette to dispense 40 microliters of sterile PBS along the side of the wells, and allow the PBS to distribute across the top of the agar bacterial microcolonies.
Prepare CARA developing reagent by mixing 5 milligrams of resazurin and 50 milliliters of 5% Tween-80 in PBS. Add 50 microliters of freshly prepared CARA developing reagent to each well of the CARA microplate. Then, rock the plates back and forth a few times to help distribute the reagent across the agar and bacterial mat in each well. Place the plates into a resealable plastic bag, and incubate at 37 degrees Celsius for at least 30 minutes for M. smegmatis.
Prior to reading fluorescence, place CARA microplates in a biosafety hood for 15 minutes at room temperature with the lids removed. When using BSL3 spectrophotometers outside of a biosafety cabinet, adhere an optical quality PCR sticker over the plate, and use a soft paper towel to seal tightly by pressing gently on the sticker surface. Determine the fluorescence via top read with excitation at 530 nanometers and emission at 590 nanometers.