オーストラリア原子科学技術学会のサポートが認められています。 TCKはAINSE賞を受賞しました。エピ医学研究所は、国立保健オーストラリアの医学研究評議会（566559）でサポートされています。 HRは、オーストラリアの大学院とBakerIDI明るい火花の賞でサポートされています。この作品は、オーストラリア政府の共同研究センタープログラムの下で設立された、サポートされている医学開発株式会社（CRC - BID）、のためのCRCによって運営されている。 COは、オーストラリアの大学院賞とCRC - BIDの補足奨学金受取人です。

1。細胞培養と実験セットアップ<ol><li>ヒトケラチノサイトは、L -グルタミン（2mM）を、上皮成長因子（5 ngのを補充したケラチノサイト- SFM（K - SFM）培地（GIBCO、無血清培地）15mLを含む75センチメートル2組織培養フラスコの単層として維持されています/ mL）を、ウシ下垂体抽出物（40μg/ mL）を、20 mg / mLのゲンタマイシン。細胞は37℃で加湿5％CO 2環境下で栽培されています</li><li>細胞は、K - SFM培地5 mLを含む12 × 25cm 2の組織培養フラスコに播種されています。 </li></ol>単一の細胞懸濁液をトリプシン処理によって調製される。細胞をリン酸緩衝生理食塩水で洗浄し、5〜10分間0.05％トリプシン/ EDTA溶液と共にインキュベートされています。細胞は丸くし、約30％が切り離されているになるために起動すると、10％ウシ胎児血清を含むダルベッコ改変イーグル培地の3倍量のトリプシンを中和するために追加されます。細胞は（20回）ピペッティングによって切り離されている。細胞を、血球計を使用してカウントされます。 適切な細胞数は細胞株の倍加時間（人間FEP - 1811ケラチノサイトで約20時間）に応じて播種されています。目的は、実験の日に〜90％コンフルエントに（フラスコあたり〜10 6細胞）を達成することです。 12フラスコから成る実験では約4時間で完了することができる、単一のクローン原性アッセイ（6非照射コントロールと6つの照射フラスコ）に最適です。 2。治療と照射<ol><li>関連する放射線修飾化合物と適当な時間のために、細胞を扱い、電離放射線γ線またはX線のいずれかに細胞をさらす。 </li></ol>通常6フラスコは、コロニー形成率（未処理）と薬剤のみコントロールとして機能する。他の六フラスコが照射されています。 1時間、水溶性天然抗酸化製剤、（20μg/ mLのための代表的データを以下に示す;整合栄養補助食品国際、スプリングヒル、テネシー州、米国CPF）この例ではヒトケラチノサイトはCinnulin PFの様々な濃度で処理されています37℃細胞は、137 Csのソース（; Nordionインターナショナル、カナダ、ON、1.6 Gyの/分Gammacell 1000年エリート照射器）を使用して、4 Gyで照射されています。 3。メッキ<ol><li>前述のように治療後に、単一の細胞懸濁液が得られる。 </li><li>各サンプル中の細胞数を血球計算板を用いて慎重にカウントし、希釈されるように適切な細胞数は、ペトリ皿（5つは15mmの皿にそれぞれの複製）に播種されています。 プレート当たり種子するセルの数を決定する際にはコロニー形成率および/または生存率が期待されるべきである。 150コロニー - 目的は、20の間の範囲を達成することです。 </li>ペトリ皿は、加湿プラスチック製のクローニングボックスに配置し、℃でコロニー形成のために37℃、5％CO 2環境下でインキュベートする。 </ol>コロニー形成のためのインキュベーション時間は、異なる細胞株では1〜3週間で変化する、それは時間が少なくとも6つの細胞分裂と同等でなければならないことが受け入れられている。この例では、ヒトケラチノサイトの制御料理は、8日が50以上のセルで構成される十分な大きさのクローンを形成する必要があります。 4。コロニーを固定と染色ドラフト内で、次の手順を実行します。 <ol><li>ゆっくりと吸引によってプレートの各々からメディアを削除する。 </li><li> 5mLの0.9％生理食塩水で各プレートを洗浄してください。 </li><li> 15〜30分間5 mLを10％中性緩衝ホルマリン液でコロニーを修正。 </li><li> 30〜60分間のdH 2 Oで希釈した5 mLの0.01％（w / v）のクリスタルバイオレットで染色。 </li><li>のdH 2 Oで過剰クリスタルバイオレットを洗い、皿が乾燥することができます。 </li></ol> 5。コロニー計数実体顕微鏡<ol><li> 50以上の個々の細胞を含むコロニーを実体顕微鏡を使用してカウントされます。 </li></ol>イメージングソフトウェアを使用して、デジタルイメージングとカウント<ol><li>コロニーのデジタル画像は、カメラやスキャン装置を用いて得られ</li><li>コロニーは、下記のように画像解析ソフトウェアパッケージを使用してカウントされます。 </li></ol> ImageJの（フィジーバージョン1.44A）を使用して細胞計数<ol><li>フィジーでの画像ファイルを開いて、ファイルにアクセスしてください - &gt;開く。 </li><li> &gt;調整...-&gt;しきい値 - 必要な8ビットフォーマットに画像を変換する場合、画像にアクセスしてください。 </li><li>唯一のコロニーが検出されるように非特異的なバックグラウンドのレベルを低減するためのしきい値を調整します。 </li><li>次のコマンドを使用してコロニーをカウント：プロセスに行く - &gt;バイナリ - &gt;最大値を検索。 </li></ol>この画像形式の場合は、ノイズ耐性は0に設定することができます。その明るい背景のオプションがチェックされていることを確認し、すべての細胞コロニーが正しく登録されていることを確認するために検出された最大値をプレビューします。

<ol>
	<li>Puck, T. T., Marcus, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+of+x-rays+on+mammalian+cells.">Action of x-rays on mammalian cells.</a> J Exp Med. 103, 653-666 (1956).</li><li>Hurlin, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+human+papillomavirus+type+18-immortalized+human+keratinocytes+to+a+malignant+phenotype.">Progression of human papillomavirus type 18-immortalized human keratinocytes to a malignant phenotype.</a> Proc Natl Acad Sci U S A. 88, 570-574 (1991).</li></ol>

利害の衝突は宣言されません。

この例では、人間のFEP - 1811ケラチノサイトは、最大100μg/ mLのCPFへの種々の濃度で処理した、データは37℃で1時間に20μg/ mLのCPF℃であるために示されています治療後の細胞は、137 Csのソース（; Nordionインターナショナル、カナダ、ON、1.6 Gyの/分Gammacell 1000年エリート照射器）を使用して、4 Gyで照射した。制御のために未処理と薬剤のみの治療法100個の細胞は、それぞれのペトリ皿に播...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Keratinocyte-serum free medium</td>
<td>Growth medium</td>
<td>Invitrogen</td>
<td>17005042</td>
<td>Supplemented with 2mM L-glutamine, 20&#x03BC;g/ml gentamicin, epidermal growth factor, bovine pituitary extract.
</td>
</tr>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15400-054</td>
<td>0.05% trypsin / EDTA to detach adherent cells; 10 x stock diluted in PBS w/o Ca2+/Mg2+.</td>
</tr>
<tr>
<td>Dulbecco&#x2019;s modified essential medium</td>
<td>Growth medium</td>
<td>Invitrogen</td>
<td>11885-084</td>
<td>Supplemented with 10% FBS and 20&mu;g/ml gentamicin; used to neutralise trypsin/EDTA.</td>
</tr>
<tr>
<td>0.09% Saline</td>
<td>Solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td> Used to wash colonies.</td>
</tr>
<tr>
<td>10% Neutral buffered formalin solution</td>
<td>Solution</td>
<td>Sigma-Aldrich</td>
<td>HT501128</td>
<td>~4% formaldehyde; used to fix colonies.</td>
</tr>
<tr>
<td>Crystal violet</td>
<td>Powder</td>
<td>SPI-Chem</td>
<td>02577-MB</td>
<td>0.01% crystal violet solution, prepared in dH2O, used to stain colonies.</td>
</tr>
<tr>
<td>Gammacell 1000 elite irradiator</td>
<td>&nbsp;</td>
<td>Nordion International Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Petri dishes</td>
<td>60 x 15 mm</td>
<td>Falcon</td>
<td>353002</td>
<td>&nbsp;</td>
<tr>
<td>Haemocytometer</td>
<td>&nbsp;</td>
<td>Hawksley; Medical and Laboratory Equipment</td>
<td>AC1000</td>
<td>&nbsp;</td>
<tr>
<td>Cloning box</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Plastic box with holes punched at opposite sides of lid; for storing petri dishes during prolonged incubation for colony formation.</td>
</tr>
<tr>
<td>Stereomicroscope</td>
<td>Type 102</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>Used to count colonies consisting of &gt;50 cells.</td>
</tr>		</tbody>
		</table>
		</div>

clonogenic assay: adherent cells

クローン原性（またはコロニー形成）アッセイは、50年以上のために設立されました。テクニックを記述したオリジナルの論文は1956年1に掲載されました。離れて方法を文書化するから、最初の画期的な研究は、培養1のX線照射した哺乳類（HeLa細胞）細胞の最初の放射線線量応答曲線を生成する。 control未処理の細胞とそのような暴露などの様々な治療を受けた細胞の間で、基本的に、クローン原性アッセイは、生殖生存率の違い（50以上の細胞のコロニーを形成する単一のセル、すなわち子孫を生成する細胞の能力）のアセスメントが可能に電離放射線、様々な化学化合物（例えば、細胞毒性薬）や他の場合における遺伝子操作へ。アッセイは、放射線生物学で最も広く受け入れられている技術となっていると広く異なる細胞株の放射線感受性を評価するために使用されています。さらに、クローン原性アッセイは、一般的に化合物を変更する放射線の有効性を監視するために、異なる細胞株のコロニー形成能力、に細胞毒性薬と他の抗がん治療薬の効果を決定するために使用されます。接着細胞株を使用する典型的なクローン原性生存実験ではコロニーを固定し、染色つの異なるコンポーネントが、1）組織培養フラスコで細胞単層の治療、2）単一の細胞懸濁液の調製とペトリ皿と3に適切な数の細胞をプレーティング）を含む関連する潜伏期間の後、これは細胞株によっては、1〜3週間から範囲にすることができます。ここでは、不死化したヒトケラチノサイト細胞株（FEP - 1811）2の使用で接着性細胞株をクローン原性アッセイを行うための一般的な手順を示しています。また、私たちの目的は、放射線に対する細胞の曝露後のコロニー形成率と生存率の分数の計算を含むクローン原性アッセイの共通の特徴を記述するために、そして天然の抗酸化製剤の使用と放射線応答の変更を例示することがあります。

Watch this Scientific Journal Video about Clonogenic Assay: Adherent Cells at JoVE.com

Clonogenic Assay: Adherent Cells

生殖可能性を評価するためのクローン原性アッセイの適用は50年以上のために設立されました。ここでは、接着細胞とクローン原性アッセイを行うための一般的な手順を示しています。

クローン原性アッセイ：付着細胞

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

Research

JoVE Journal

Biology

47.6K ビュー.  The Alfred Medical Research and Education Precinct. 生殖可能性を評価するためのクローン原性アッセイの適用は50年以上のために設立されました。ここでは、接着細胞とクローン原性アッセイを行うための一般的な手順を示しています。

ビデオ: Clonogenic Assay: Adherent Cells

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

接着細胞培養用の酸素挿入の作製と動作

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...

生物学

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

接着および非接着性の哺乳動物細胞の in situ細胞下分画で

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

マウスの主栄養膜細胞と絨毛浸潤アッセイの分離

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

3&#39;UTR -レポーターコンストラクトとマイクロRNAを使用してLightSwitchルシフェラーゼアッセイシステムによるヒトマイクロRNAの識別対象は付着細胞へのミミック

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

間葉転換を誘導し、上皮の分析

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

ギャップ結合審査のための機能アッセイ;インジウム - スズ酸化物に対する接着した細胞のエレクトロポレーション

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

ゼブラフィッシュにおける腎毒素マイクロインジェクションは急性腎障害をモデルに

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

動的細胞突起におけるソフトウェア支援トラッキングのためのグラフィカルユーザインタフェース

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

レンズ無料のビデオ顕微鏡、付着性細胞培養の定量的解析

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated &#945; subunit (HIF-1&#945;) and constitutively expressed &#946; subunit (HIF-1&#946;) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis. The identification of HIF-1 interacting proteins is key to the understanding of the hypoxia signaling pathway. Besides the regulation of HIF-1&#945; stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1&#945; and ARNT. Notably, most of the current methods used to study such protein-protein interactions (PPIs) are based on systems where protein levels are artificially increased through protein overexpression. Protein overexpression often leads to non-physiological results arising from temporal and spatial artifacts. Here we describe a modified co-immunoprecipitation protocol following hypoxia treatment using endogenous nuclear proteins, and as a proof of concept, to show the interaction between HIF-1&#945; and ARNT. In this protocol, the hypoxic cells were harvested under hypoxic conditions and the Dulbecco's Phosphate-Buffered Saline (DPBS) wash buffer was also pre-equilibrated to hypoxic conditions before usage to mitigate protein degradation or protein complex dissociation during reoxygenation. In addition, the nuclear fractions were subsequently extracted to concentrate and stabilize endogenous nuclear proteins and avoid possible spurious results often seen during protein overexpression. This protocol can be used to demonstrate endogenous and native interactions between transcription factors and transcriptional co-regulators under hypoxic conditions.

Here we describe a co-immunoprecipitation protocol to study protein-protein interactions between endogenous nuclear proteins under hypoxic conditions. This method is suitable for demonstration of the interactions between transcription factors and transcriptional co-regulators at hypoxia.

低酸素の条件の下で培養された細胞から内因性の核蛋白質を用いた Co 免疫沈降試験

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. ...