Sigma - Aldrich社によって資金を供給

1。マイコプラズマ検出<ol><li>細胞の培養上清を直接テストすることができるまたはサンプルは、後日使用する目的で作成できます。の準備をするために、後95℃で滅菌増幅管とインキュベートで上清の代わりに100μlのを使用して℃で5分間。一度これは、サンプルが最大1週間2〜8℃で保存することができます完了です。ちょうど前のペレットのサンプルの短時間の遠心（5秒）、任意の細胞破片を実行する。 </li><li> PCR用サンプルを準備するには、反応に必要なジャンプスタートTaq DNAポリメラーゼ/再水和バッファの総量を決定する。我々は、合計5つの反応を準備されます。五反応はTaqの2.5μlおよび再水和のバッファの114.5μl必要になります。これはサンプルの反応とポジティブコントロールのための補水のバッファの負の制御に加え、24.5μlあたりの水分補給のバッファの反応と22.5μlあたりTaqの一単位の最小値が含まれます。反応あたりのDNAポリメラーゼの1単位は、補水のバッファの適切な音量に追加する必要があります。これは、使用されるTaqポリメラーゼによって異なります。 </li><li>きれいなマイクロ遠心チューブにTaq DNAポリメラーゼの計算されたボリュームを配置し、補水のバッファの計算量で従ってください。 DNAポリメラーゼ/水分補給のバッファは、チューブをタッピングし穏やかに混合されるべきである。この混合物をボルテックスしてはいけません。 </li><li>ネガティブコントロールとサンプルを準備するには、キットに付属の透明な反応チューブを使用してください。キットに含まれている反応管は既にnuclueotides、プライマーと内部コントロールDNAが含まれています。前の手順で準備したジャンプスタートTaq DNAポリメラーゼ/膨潤バッファーミックス23μlを、ネガティブコントロールとサンプルチューブの各々に配置する必要があります。ネガティブコントロールするDNAフリー水を2μlを加え、サンプルチューブとラベルの各サンプル2μlを加える。チューブをフリックでコンテンツを混在させること。内容をボルテックスしてはいけません。 </li><li>準備するにはポジティブコントロールはキットに付属のピンクの反応チューブを使用してください。キットに含まれている反応管は既にnuclueotides、プライマーと内部コントロールDNAが含まれています。反応管およびラベルに、前の手順で準備した、ジャンプスタートTaq DNAポリメラーゼ/膨潤バッファーミックス25μlを添加する。チューブをフリックでコンテンツを混在させること。内容をボルテックスしてはいけません。 5分間室温でネガティブコントロール、ポジティブコントロールおよびサンプルチューブをインキュベートする。 </li><li> PCRの場所にするために、サンプルは、サーマルサイクラーに配置する必要があります。 JumpStartのTaqを使用する場合、アクティベーション手順は必須ではありません。サーマルサイクラーでの反応チューブを置きます。サイクルは次のように設定する必要があります：94℃30秒、55℃30秒と40秒72℃のためのC。これらのサイクルを40回実行されます。 </li><li> PCRのサイクルが完了したらサンプルをサーマルサイクラーからそれらを削除し、氷に入れて、4〜8 ° Cまで冷却する必要があります。試料が冷却されているとき、彼らは電気泳動用アガロースゲルにロードする必要があります。彼らはすでに反応管に存在する限りローディングゲルと染料は不要です。直接、別の車線に各PCRに8μlをロードする。 3.0センチメートル - 2.5の移行後に電気泳動を停止します。 </li></ol> 2。代表的な結果ネガティブコントロールは、約481 bpのバンドが表示されるはずです。これは、ビデオの先頭に表示されている電気泳動ゲル上でネガティブコントロールの列で見ることができます。 481 bpのネガティブコントロールのバンドがない場合は、ポリメラーゼの活性がsufficienttaqが使用されていないことを示す良い指標が十分に敏感ではなかったです。 マイコプラズマ陽性検体は、270の範囲でバンドを± 8塩基が表示されます。バンドは、高度に汚染されたサンプルの重いと軽く汚染されたサンプルのかすかになります。強度の変化は、ビデオの先頭に表示されている電気泳動ゲル上の正のサンプルの列で見ることができます。すべてのサンプルは、PCRが期待どおりに発生したことを示すために内部ネガティブコントロールが含まれています。それは高度に汚染された試料の内部統制が存在しないことを確認するのが普通です。 あなたのサンプルの負の範囲で正の範囲またはバンドのバンドがない場合、これはあなたのサンプルが阻害されていることを示す良い指標です。サンプルが禁止されている場合はPCRの阻害物質は、DNAの抽出を行うことにより除去することができます。 <img alt="図1" src="/files/ftp_upload/3057/3057fig1.jpg" /> 図1関連するアンプリコンのバンド：PCRによるマイコプラズマ検出の結果が表示されます。レーン2は、約481 bpのバンドが表示されるはず陰性コントロール、です。 481 bpのネガティブコントロールのバンドがない場合は、使用されるTaqポリメラーゼが十分に敏感ではなかったことを示す良い指標です。 6 - マイコプラズマ陽性サンプルは、レーン3に示すように270 + 8 bpの範囲でバンドが表示されます。バンドは、HEAになりますVY高度に汚染された試料（レーン6）と軽く汚染された試料（レーン4）のためのかすかなため。すべてのサンプルは、PCRが期待どおりに発生したことを示すために内部陰性コントロール（481 bp）を含んでいます。それは高度に汚染された試料の内部統制が存在しないことを確認するのが普通です。

<ol>
	<li>Wirth, M., Hauser, H., Drexler, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+and+Sensitivity+of+Polymerase+Chain+Reaction+(PCR)+in+Comparison+with+Other+Methods+for+the+Detection+of+Mycoplasma+Contamination+in+Cell+Lines.">Specificity and Sensitivity of Polymerase Chain Reaction (PCR) in Comparison with Other Methods for the Detection of Mycoplasma Contamination in Cell Lines.</a> Journal of Immunological Methods. 164, 91-100 (1993).</li><li>Kong, H., Volokhov, D. V., George, J., Ikonomi, P., Chandler, D., Anderson, C., Chizhikov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplification+of+Cell+Culture+Enrichment+for+Improving+the+Sensitivity+of+Mycoplasma+Detection+Methods+Based+on+Nucleic+Acid+Amplification+Technology+(NAT).">Amplification of Cell Culture Enrichment for Improving the Sensitivity of Mycoplasma Detection Methods Based on Nucleic Acid Amplification Technology (NAT).</a> Applied Microbiology and Biotechnology. 77, 223-232 (2007).</li><li>Volokhov, D. V., Graham, L. J., Borson, K. A., Chizhikov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+Testing+of+Cell+Substrates+and+Biologics:+Review+of+Alternative+Non-Microbiological+Techniques.">Mycoplasma Testing of Cell Substrates and Biologics: Review of Alternative Non-Microbiological Techniques.</a> Molecular and Cellular Probes. , (2011).</li></ol>

著者は、この記事で使用される試薬やツールを生成するシグマアルドリッチの従業員です。

のでキットの極端な感度で、正しく実行時キットは、サンプルがマイコプラズマ汚染があるかどうかを示す正確な結果が得られるはずです。このキットは、JumpStart Taq DNAポリメラーゼの使用に最適化されています。他のtaqsはサンプルを準備する際に使用されることがありますが、481 bpの内部統制のバンドは、代替Taqの適切な感度を示すために存在している必要があります。それは、汚染のレベルに応じて様々な密度を示すために正のバンドは可能です。汚れが多い試料での内部統制のバンドの有無を観察することが可能です。汚染を防ぐためにサンプルを準備する際の注意と適切なテクニックを使用すると、適切なため非常に重要です。見張りマイコプラズマPCR検出キットを用いて検出。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td>見張りマイコプラズマPCR検出キット</td><td>シグマアルドリッチ</td><td> MP0035 </td><td> </td></tr><tr><td> JumpStartのTaq DNAポリメラーゼ</td><td>シグマアルドリッチ</td><td> D9307 </td><td> </td></tr><tr><td> GenElute血液ゲノムDNAキット</td><td>シグマアルドリッチ</td><td> MP0030 </td><td> </td></tr><tr><td>ヌンク増幅管</td><td>シグマアルドリッチ</td><td> T0447 </td><td> </td></tr></tbody></table>

optimized pcr-based detection of mycoplasma

汚染のない細胞株の維持は、細胞ベースの研究に不可欠です。最大の汚染物質の懸念の中でマイコプラズマ汚染です。マイコプラズマは、通常、汚染された細胞を殺すことはありませんが、彼らは検出することが困難であり、新陳代謝の変化を含む培養細胞、、上の様々な効果を引き起こす可能性増殖と染色体異常を遅らせた。一言で言えば、マイコプラズマ汚染がライフサイエンス研究のための正確なデータを提供することで、これらの細胞株の価値が損なわれます。 実験室におけるマイコプラズマ汚染の源は非常に完全に制御することが挑戦しています。特定のマイコプラズマ種が人間の皮膚に見られるように、彼らは貧しい無菌操作を介して導入することができます。さらに、彼らはそのようなウシ胎児血清などの汚染されたサプリメントから来ることができる、そして最も重要な他の汚染された細胞培養物から。一度マイコプラズマが文化を汚染、それはすぐに研究室の他の地域を汚染して広げることができます。良い無菌操作などの検査室の安全基準を厳守が鍵であり、マイコプラズマのためのルーチン検査は、非常にマイコプラズマ汚染の成功を制御することをお勧めします。 マイコプラズマのPCRベースの検出は、ルーチン細胞株の維持のための非常にポピュラーな方法となっている。 PCRベースの検出方法は非常に敏感であり、研究者は、それが微生物学的手法を用いて必要な時間と比較して検出されると、汚染を分離し、除去するために迅速に対応できるように迅速な結果を、提供することができます。見張りマイコプラズマPCR検出キットは、ウェルあたりわずか2ゲノムの検出限界で、高感度です。高度に特異的なJumpStartのTaq DNAポリメラーゼと独自のプライマー設計を活かし、偽陽性が大幅に削減されます。便利な8連チューブのフォーマット、dNTPでプレコートされたストリップ、および関連するプライマーは、細胞株の大規模なコレクションを、お客様のニーズを満たすために、スループットの向上に役立ちます。 キットの極端な感度を考えると、細心の注意を試料と試薬の不注意による汚染を防ぐために注意しなければなりません。我々は実証ステップバイステップのプロトコルは、信頼性の高いマイコプラズマ検出に必要な予防措置や慣行を強調しています。我々はまた、表示と典型的な結果とその解釈について説明します。私たちの目標は、目を光らせマイコプラズマPCR検出キットを用いて研究者の成功を確実にするためです。

Watch this Scientific Journal Video about Optimized PCR-based Detection of Mycoplasma at JoVE.com

Optimized PCR-based Detection of Mycoplasma

見張りマイコプラズマPCR検出キットは、細胞培養と生物製剤由来の他の細胞培養で、マイコプラズマAcholeplasma、およびウレアプラズマ汚染の検出に最も感度のための選択の方法として確立されて、ポリメラーゼ連鎖反応（PCR）を利用しています。

マイコプラズマの最適化されたPCRベースの検出

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

optimized-pcr-based-detection-of-mycoplasma

Research

JoVE Journal

Biology

53.5K ビュー.  Sigma-Aldrich. 見張りマイコプラズマPCR検出キットは、細胞培養と生物製剤由来の他の細胞培養で、マイコプラズマAcholeplasma、およびウレアプラズマ汚染の検出に最も感度のための選択の方法として確立されて、ポリメラーゼ連鎖反応（PCR）を利用しています。

ビデオ: Optimized PCR-based Detection of Mycoplasma

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

血管新生の研究のために最適化されたフィブリンゲルビーズアッセイ

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

生物学

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

タンパク質のユビキチン化の検出

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

ザイモグラフィーによる機能マトリックスメタロプロテイナーゼの検出

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage1. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus has been a successfully used for biological control of fruit flies in multiple tropical regions2-4. One impediment to the wide use of F. arisanus for fruit fly control is that it is difficult to establish a stable laboratory colony5-9. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus10-12. There is significant interest in F. arisanus biology13,14, especially regarding its ability to colonize a wide variety of Tephritid hosts14-17; interest is especially driven by the alarming spread of Bactrocera fruit fly pests to new continents in the last decade18. Further research on F. arisanus and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus based on a previously described approach12. The method we describe here allows rearing of F. arisanus in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

子育てに最適化されたプロトコル Fopius arisanus、Tephritid果実の寄生ハエ

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

の野生型と観賞用品種を区別するPCRベースのジェノタイピング法チガヤ</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

イメージングと免疫組織化学のための網膜ホールマウントの準備のために最適化されたプロトコル

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta replacement. The electrospun vascular grafts often possess relatively small pores, which limit cell infiltration into the grafts and hinder the regeneration and remodeling of the neo-arteries. In this study, PCL vascular grafts with thicker fibers (5 - 6 &#181;m) and larger pores (~30 &#181;m) were fabricated by using a modified processing technique. The long-term performance of the graft was evaluated by implantation in a rat abdominal aorta model. Ultrasound analysis showed that the grafts remained patent without aneurysm or stenosis occurring even after 12 months of implantation. Macroporous structure improved the cell ingrowth and thus promoted tissue regenerated at 3 months. More importantly, there was no sign of adverse remodeling, such as calcification within the graft wall after 12 months. Therefore, electrospun PCL vascular grafts with modified macroporous processing hold potential to be an artery substitute for long-term implantation.

Here, we present a modified electrospinning method to fabricate PCL vascular grafts with thick fibers and large pores, and describe a protocol to evaluate the in vivo performance in a rat model of abdominal aorta replacement.

エレクトロスピニング ラットモデルにおける最適化された構造を有する人工血管の移植

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.

スクラッチのアッセイの in Vitro細胞遊走の自動光学カメラのテストのために最適化されました。

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...

Simple Detection of Primary Cilia by Immunofluorescence

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to mitosis. Primary cilia can be visualized with highly sophisticated methods, including transmission electron microscopy, 3D imaging, or using software for the automatic detection of primary cilia. However, immunofluorescent staining of primary cilia is needed to perform these methods. This publication describes a protocol for the easy detection of primary cilia in vitro by staining acetylated alpha tubulin (axoneme) and gamma tubulin (basal body). This immunofluorescent staining protocol is relatively simple and results in high-quality images. The present protocol describes how four cell lines (C2C12, MEF, NHLF, and skin fibroblasts) expressing primary cilia were fixed, immunostained, and imaged with a fluorescent or confocal microscope.

Primary cilia are extracellular structures associated with the centriole. Primary cilia detection by immunofluorescent staining is a relatively simple procedure that results in extremely high-quality images. In this protocol, fibroblasts expressing primary cilia were fixed, immunostained, and imaged in a fluorescent or confocal microscope.

免疫蛍光による原発性繊毛の簡易検出

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to ...

Automated Detection and Analysis of Exocytosis

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic events in cell culture. To perform an unbiased, efficient identification and analysis of such events, a computer-vision based approach was developed and implemented in MATLAB. The analysis pipeline consists of a cell segmentation and exocytic-event identification algorithm. The computer-vision approach includes tools for investigating multiple parameters of single events, including the half-life of fluorescence decay and peak &#916;F/F, as well as whole-cell analysis of the frequency of exocytosis. These and other parameters of fusion are used in a classification approach to distinguish distinct fusion modes. Here a newly built GUI performs the analysis pipeline from start to finish. Further adaptation of Ripley&#39;s K function in R Studio is used to distinguish between clustered, dispersed, or random occurrence of fusion events in both space and time.

We developed automated computer vision software to detect exocytic events marked by pH-sensitive fluorescent probes. Here, we demonstrate the use of a graphical user interface and RStudio to detect fusion events, analyze and display spatiotemporal parameters of fusion, and classify events into distinct fusion modes.

エキソサイトーシスの自動検出と解析

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic ...