著者らは、マウスNgn3 - EGFP胚性幹細胞株、夫人ルーシーブラウンと細胞選別の支援のための分析サイトメトリーコアのプレゼントのために博士クラウスケストナーに感謝したいと思います。この作品は、MWとLSに授与訓練助成金HTKに授与NIH助成金R21DK069997とR01DK081587によってと再生医療（CIRM）のためのカリフォルニア工科大学から一部で賄われていた

<ol>
	<li>Ku, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Committing+embryonic+stem+cells+to+early+endocrine+pancreas+in+vitro.">Committing embryonic stem cells to early endocrine pancreas in vitro.</a> Stem cells. 22, 1205-1217 (2004).</li><li>Ku, H. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage-specific+morphogenesis+in+the+developing+pancreas:+role+of+mesenchymal+factors.">Lineage-specific morphogenesis in the developing pancreas: role of mesenchymal factors.</a> Development. 122, 439-447 (1996).</li><li>Miralles, F., Battelino, T., Czernichow, P., Scharfmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+plays+a+key+role+in+morphogenesis+of+the+pancreatic+islets+of+Langerhans+by+controlling+the+activity+of+the+matrix+metalloproteinase+MMP-2.">TGF-beta plays a key role in morphogenesis of the pancreatic islets of Langerhans by controlling the activity of the matrix metalloproteinase MMP-2.</a> J. Cell. Biol. 143, 827-836 (1998).</li><li>Bonner-Weir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cultivation+of+human+islets+from+expanded+ductal+tissue.">In vitro cultivation of human islets from expanded ductal tissue.</a> Proc. Natl. Acad. Sci. U. S. A. 97, 7999-8004 (2000).</li><li>Gao, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+endocrine+progenitor+cells+and+critical+factors+for+their+differentiation+in+human+adult+pancreatic+cell+culture.">Characterization of endocrine progenitor cells and critical factors for their differentiation in human adult pancreatic cell culture.</a> Diabetes. 52, 2007-2015 (2003).</li><li>Gao, R., Ustinov, J., Korsgren, O., Otonkoski, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+neogenesis+of+human+islets+reflects+the+plasticity+of+differentiated+human+pancreatic+cells.">In vitro neogenesis of human islets reflects the plasticity of differentiated human pancreatic cells.</a> Diabetologia. 48, 2296-2304 (2005).</li><li>Suarez-Pinzon, W. L., Lakey, J. R., Brand, S. J., Rabinovitch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+therapy+with+epidermal+growth+factor+and+gastrin+induces+neogenesis+of+human+islet+beta-cells+from+pancreatic+duct+cells+and+an+increase+in+functional+beta-cell+mass.">Combination therapy with epidermal growth factor and gastrin induces neogenesis of human islet beta-cells from pancreatic duct cells and an increase in functional beta-cell mass.</a> J. Clin. Endocrinol. Metab. 90, 3401-3409 (2005).</li><li>Hao, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-cell+differentiation+from+nonendocrine+epithelial+cells+of+the+adult+human+pancreas.">Beta-cell differentiation from nonendocrine epithelial cells of the adult human pancreas.</a> Nature Medicine. 12, 310-316 (2006).</li><li>Ku, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+pancreatic+progenitor+cells--recent+studies.">Minireview: pancreatic progenitor cells--recent studies.</a> Endocrinology. 149, 4312-4316 (2008).</li><li>Seaberg, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+identification+of+multipotent+precursors+from+adult+mouse+pancreas+that+generate+neural+and+pancreatic+lineages.">Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages.</a> Nature Biotechnology. 22, 1115-1124 (2004).</li><li>Rovira, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+centroacinar/terminal+ductal+progenitor+cells+in+adult+mouse+pancreas.">Isolation and characterization of centroacinar/terminal ductal progenitor cells in adult mouse pancreas.</a> Proc. Natl. Acad. Sci. U. S. A. 107, 75-80 (2010).</li></ol>

我々は、開示することは何もない。

個々の膵前駆細胞の定量的および機能的な評価を可能にするコロニーアッセイは、非常に膵臓の幹細胞や前駆細胞生物学の研究の進展を妨げている、これまでのところ限られている。コロニーアッセイの本質は、前駆細胞の機能と系統の電位が観察できるように、本質的な前駆細胞の能力ではなく、単にそれらの表現型を測定することです。また、特定の集団に前駆細胞の数を決定することができる定量的なツールです。造血幹細胞の分野で、コロニーアッセイが同定に重要な役割を果たしただけでなく、成長因子の要件、相互作用、およびこれらの細胞の系譜のコミットメントを支配するメカニズムを解読している。膵前駆細胞の生物学の分野6、膵臓の臓器7 、8またはバルク文化9月13日考案されているが、しかしながら、このようなアッセイは、生物学的クエスティに対処していない単一細胞レベルで作成されている。単一細胞分析することなく、複数の潜在的な膵臓の幹/前駆細胞の存在が明確に立証することはできません。14 ここでは、インスリンを発現するコロニー形成前駆細胞が三次元、マトリゲルを含む、半固体培養系を用いて測定することができます。見せる懸濁培養系15,16を単一の成人の膵臓の前駆細胞が増殖し、&quot;pancreatospheres&quot;に分化することを可能にするどの開発されているが、我々のコロニーアッセイでは、付加的な利点を提供します。最初に、私たちの半固体培養のメディアシステムは、最大24ウェルあたり500μLの培地25,000ソートされた細胞へのメッキ線形範囲で得られたコロニーの形成を維持しながら。2は対照的に、サスペンションのpancreatosphereアッセイは、単一の使用を必要とすることが可能セルソーターによる細胞の成膜方法は、単一細胞分析を可能にするため、文化、メディアとインキュベータスペースの大きなボリュームに必要です。したがって、我々のコロニーアッセイでは、異なる多数の細胞が前駆細胞の活動のために同時に比較する必要がある場合に実施するのは比較的簡単で費用対効果です。第二に、私たちの半固形培地では増殖と分化の両方が同じウェルで発生することができる細胞外マトリックス成分と成長因子の均一な分布を、可​​能です。対照的に、それは、懸濁培養中の細胞にこのような環境、特に行列を、提供することより困難である。我々は2つ​​の前駆細胞がランダムにお互いとフォームつのコロニーの近くに配置することができるように私たちの方法でそれは決定的に、各コロニーは単一の細胞から派生していることを証明することはできないことを認識しています。しかし、この問題は、単一細胞操作を用いた二次分析によって克服することができます。ソートされた単一細胞の直接手摘みでは決定的に各コロニーの単セルの原点を示すことができる。 要約では、プロのインスリン発現コロニー形成活性ここに記載さ半固形培地でgenitorsは、単一細胞レベルでの膵前駆細胞の力学的研究のためのユニークな機会を提供しています。例えば、前の報告書で我々は細胞外マトリックスを示唆し2、その培養液へのマトリゲル、細胞外マトリックス蛋白質の粗混合物を添加することはインスリン発現Ngn3 - EGFP +細胞からのコロニーの形成に必要な発見です。それらの前駆細胞の重要な。このコロニーアッセイを使用して、それは現在、さらに細かく分析し、そのような活動のための特定のマトリックスの要件を定義することが可能となります。マウス胚性幹細胞由来の前駆細胞を研究に加えて、我々は現在、in vitroのアッセイ系で 、これはまた、周産期および成体マウスの膵臓と肝臓から、同様に欠けている人間の死体膵組織由来の他の特徴的なコロニーの形成をサポートするかどうかを調査しています膵島の。

<table border="1"><tbody><tr><td> 材料/試薬 </td><td> 会社 </td><td> カタログ 数 </td><td> コメント </td></tr><tr><td> 1X DMEM / F - 12（1:1） </td><td> Mediatech </td><td> 15から090 - CV </td><td></td></tr><tr><td>ウシ胎児血清</td><td>組織文化 生物学的製剤</td><td> 201 </td><td>セクションを参照してください。 1.1 </td></tr><tr><td> L -グルタミン</td><td> GIBCO </td><td> 25030-081 </td><td></td></tr><tr><td> 1XダルベッコPBS </td><td> Mediatech </td><td> 21から031 - CV </td><td></td></tr><tr><td>ウシ血清 アルブミン</td><td>シグマ</td><td> A8412 </td><td> 7.5パーセント</td></tr><tr><td>マトリゲル</td><td>ベクトン ディッキンソン</td><td> 356231 </td><td>削減成長 ファクタ</td></tr><tr><td>メチルセルロース</td><td> Sinetsu 化学物質、 東京、日本</td><td></td><td> 1500センチポイズ （高VIscosity） </td></tr><tr><td>ニコチンアミド</td><td>シグマ</td><td> N0636 </td><td></td></tr><tr><td>ヒトの 組換え アクチビンβB </td><td> R＆Dシステムズ</td><td> 659 - AB - 025 </td><td></td></tr><tr><td>エキセンディン-4 </td><td>シグマ</td><td> E7144 </td><td></td></tr><tr><td>ヒトの 組換え VEGF - </td><td> R＆Dシステムズ</td><td> 293 - VE - 010 </td><td></td></tr><tr><td> 24ウェルプレート</td><td>共演スター</td><td> 3473 </td><td>超低 添付</td></tr><tr><td> 60ミリメートルシャーレ グリッドと</td><td>ヌンク</td><td> 169558 </td><td></td></tr></tbody></table>

a quantitative assay for insulin-expressing colony-forming progenitors

膵臓の幹細胞や前駆細胞生物学の分野は、単一細胞の分析を可能にするin vitro機能と定量的なアッセイでの不足によって妨げられている。彼らは明確に個々の前駆細胞の系統の可能性を実証する決定的な方法を提供するため、単一の前駆細胞の解析は非常に重要です。メソッドは、単一の細胞から浮遊培養で&quot;pancreatospheres&quot;を生成するために考案されているが、いくつかの制限があります。最初、それは文化のメディアと空間のターンコマンドで大量の細胞の数が多い、のために単一のセルの堆積を行うために時間がかかります。結果pancreatospheresの第二、記数はpancreatosphere -開始前駆細胞の頻度が低い場合は特に、労働集約的である。第三に、pancreatosphereアッセイは、再、よく同じ文化の中で膵臓の前駆細胞の増殖と分化の両方を可能にする効率的な方法ではありませんアッセイの有用性をstricting。 これらの制限を克服するために、膵臓の前駆細胞のための半固体培地によるコロニーアッセイが開発され、本報告書で提示されます。このメソッドは、それらが増殖ならびに分化を受けるように前駆細胞が三次元空間に滞在できるように、メチルセルロースがメディアに粘度を提供するために使用されている造血コロニーアッセイ、から、既存の概念を活用します。不均一な集団からインスリンを発現するコロニー形成前駆細胞を豊かにするため、我々は急行neurogenin（NGN）3、膵内分泌前駆細胞のマーカーその細胞を利用した。マウス胚性幹（ES）強化緑色蛍光タンパク質レポーターでタグ付けさNgn3発現細胞をソートされ、ウェル当たり、多くの25000のような細胞が低添付ファイル24穴培養皿に播種した。細胞が由来次の主要コンポーネントと半固体培地の各ウェルに含まれる500μL：METhylcellulose、マトリゲル、ニコチンアミド、エキセンディン-4、アクチビンβB、および馴化培地は、マウスES細胞由来の膵臓様細胞から採取。文化8〜12日後に、独特の形態を持つインスリンを発現するコロニーが形成され、さらに各コロニーの系統構成を決定するために定量的RT - PCRとimmunoflourescent染色を用いて膵臓遺伝子発現を分析することができます。 要約すると、我々のコロニーアッセイは、不均一な細胞集団内で機能的な前駆細胞の容易な検出および定量することができます。さらに、半固体のメディアフォーマットは、in vitroで増殖し、区別するために前駆細胞を有効に、細胞に細胞外マトリックス成 ​​分と成長因子の一様なプレゼンテーションが可能になります。このコロニーアッセイは、単一細胞レベルでの膵前駆細胞の力学的研究のためのユニークな機会を提供しています。

Watch this Scientific Journal Video about A Quantitative Assay for Insulin-expressing Colony-forming Progenitors at JoVE.com

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

膵臓のような前駆細胞がインスリンを発現するコロニーへの分化を可能にする三次元のクローン原性アッセイが記載されている。単一の前駆細胞が増殖と分化するこの方法は、メチルセルロース、マトリゲルと成長因子を含有する半固体培地を利用しています<em&gt; in vitroで</em&gt;、人口で機能的な前駆細胞数の定量化を可能にする。

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-quantitative-assay-for-insulin-expressing-colony-forming-progenitors

Research

JoVE Journal

Biology

13.4K ビュー.  California State University Channel Islands. 膵臓のような前駆細胞がインスリンを発現するコロニーへの分化を可能にする三次元のクローン原性アッセイが記載されている。単一の前駆細胞が増殖と分化するこの方法は、メチルセルロース、マトリゲルと成長因子を含有する半固体培地を利用しています in vitroで、人口で機能的な前駆細胞数の定量化を可能にする。

ビデオ: A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

脂肪酸の定量的なPhosphoproteomicsは刺激を受けてサッカロマイセスセレビシエ</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

生物学

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

分離と成人ヒト内皮コロニー形成前駆細胞の大規模拡張

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

ヒト臍帯由来間葉系幹細胞（MSCs）と血管内皮コロニー形成前駆細胞（ECFCs）の単離および動物血清自由膨張

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. 
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

CD71/TER119フローサイトメトリーアッセイを用いてマウス赤血球前駆細胞の同定と解析

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

ヒト臍帯血由来血管内皮コロニー形成細胞の表現型および機能解析

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

定量的なフィットネスの分析ワークフロー

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

ペプチドエピトープの定量分析のための高スループット、MHC II結合アッセイ

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

敏感な、大規模定量的メタボロームのための戦略

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

中心体におけるタンパク質レベルの変化を測定するための定量的免疫蛍光アッセイ

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

内部陽性対照との定量的リコンビナーゼポリメラーゼ増幅アッセイの開発

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...