TMSは、この研究の間にハーラン·トラスト·フェローシップ（GWU）によってサポートされていました。フィールドコレクションと繁殖L.のための資金蛭とE. delphiniiは JTLにNSFの助成DEB 0642438によって提供されました。

1。材料の準備<ol><li>ホウケイ酸ガラスキャピラリーチューブとマイクロピペットプラーを使用して針（1-2μmの先端と3〜4mmのテーパー）を準備します。機器設定：ランプ：561;速度：20;熱：560;ディレイ：1;プル：100;圧力：500。 </li><li>ウシ胎児血清（FBS）の10％および抗凝固バッファの20％（98 mMのNaOHを用い、186のNaCl、1.7mMのEDTAおよび41 mMクエン酸、pH 4.5を補ったグレース培地（GM）の60％捕集溶液を調製）。上記溶液を滅菌条件下で新たに調製し、常に氷上に維持されています。 </li><li> GMの90％FBSを10％ウシ：インキュベーション緩衝液を準備します。このソリューションは、新たに調製し、常に氷上に維持されています。 </li><li>少なくとも15μlの合計×収集·インキュベーション·ソリューションの両方の昆虫の数を準備します。 </li></ol> 2。コレクション·ソリューションとの毛虫の注入<ol><li>最後から二番目のプラグを麻酔血球抽出に先立って20分間氷上でそれらを冷却することによりタールの幼虫。 </li><li> 20ミリリットルのガラス製注射器（ 図2）に接続されているチューブにきれいな毛管針（16ゲージ）の鈍端を取り付けます。収集ソリューションは、ラボ·ベンチで開いて、ゆっくりと緩衝液を用いてキャピラリーを埋める入ったチューブのままにしておきます。毛細血管は、通常毛虫のサイズに応じて、収集ソリューションの約10〜15μlで満たされている（毛虫合計体重の10 mg当たり約1μlの溶液=総体重の0.01％）。解剖顕微鏡の横に配置された粘土のボールの上に針を置きます。 </li><li>ペトリ皿に毛虫を置きます。昆虫は、気門が（ 図3）はっきりと見えるように、その側に配置されるべきである。 </li><li>解剖顕微鏡で見ながら、ピンセットで所定の位置に毛虫を保持し、気門の1つに針の近くに配置する場所で宗派のキューティクルは、一般的に軟質である。優しく昆虫の身体に対してキャピラリーニードルを押してください。針がキューティクルに浸透した後は、キャタピラ（ 図3）に溶液の全体積を注入します。毛虫に気泡を注入避けることが重要である。毛虫の体は注射後（ 例えば 、20％の体積の増加）膨潤するが、破裂したり、にじみ出るべきではありません。 </li><li>傷（ 図3）から漏れるのを防ぐためにバッファ背虫を配置し、氷に毛虫を返します。 </li><li>体液の抽出に使用される各毛虫の手順2.1から2.4を繰り返します。この段階では、すべての昆虫が進む前に、30分間氷上で冷却することができます。 </li></ol> 3。血液細胞の抽出<ol><li>解剖顕微鏡下でシャーレに毛虫を置きます。 </li><li>無菌scalpeを使用して毛虫の臼歯部では浅い（2-3 mm長）を切開する<html> L（ 図3）。腸や他の臓器に損傷を与えないように注意してください。目標は慎重に穿刺にキューティクルであり、個々の昆虫から血球を含有する緩衝液/血リンパミックスを収集します。 </li><li> 10μlのピペットチップで体液ミックス（約10〜20μl）を収集しながら、再び内部の組織（ 図3）を傷つけないように注意しながら、柔軟なプラスチック製のピンセットを使って静かに毛虫を絞る。血リンパは毛虫の体から搾り液滴から収集されます。毛虫の遺体を捨ててください。 </li><li>別のラベルの付いたシリコーン処理、0.5mlのマイクロ遠心チューブに、個々の毛虫から体液を採取します。 </li><li>個々の毛虫から収集された各体液試料にインキュベーション溶液の等量（約10〜20μl）を追加します。すべての回で氷の上に体液サンプルを保管してください。 </li><li>実験に用いた各毛虫の手順3.1から3.5を繰り返します。 </li></ol>血液細胞の定量LE &quot;&gt; 4。 <ol><li>体液試料および血球計数直前に培養液を混ぜる。この治療法は、血球の適切な分離を可能にし、凝集を防ぐことができます。 </li><li>血球計算板の上に、インキュベーション溶液と混合体液サンプル10μlを転送するためにクリーンなピペットチップを使用しています。 </li><li>血球計算板の中央グリッド（ 図3）の化合物の顕微鏡を使用して、大きい正方形（0.1ミリメートル：0.04ミリメートル2、深さが1の正方形の面積）の少なくとも15の血球の数をカウントし、記録します。 15正方形の血球数の総和を得るためにこれらの値を追加します。 </li><li>徹底的に蒸留、脱イオン水で洗浄瓶を用いて血球計算板とカバーのスライドを洗浄し、各サンプル間キムワイプ（キンバリークラーク）で拭いて乾かしてください。 </li><li>各体液サンプルについてステップ4.1から4.4を繰り返します。 </li><li>血リンパvolumのmlあたり血球の平均数を計算次のように各毛虫のe、： </li></ol>血球計算グリッドの各四角形の総容積：（0.04 mm 2の面積）×（0.1ミリメートルの深さ）= 0.004ミリメートル3×15乗= 0.06ミリメートル3全15カウントの正方形=総量。 （全15カウントの正方形/総量カウント15の正方形の血球の総数の合計）×2希釈係数=血球/ mm 3の数×1,000 =血球/体液のミリリットル数。

<ol>
	<li>Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+cells+and+blood+cell+development+in+the+animal+kingdom.">Blood cells and blood cell development in the animal kingdom.</a> Annu. Rev. Cell Dev. Biol. 22, 677 (2006).</li><li>Wood, W., Jacinto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+embryonic+haemocytes:+masters+of+multitasking.">Drosophila melanogaster embryonic haemocytes: masters of multitasking.</a> Nat. Rev. Mol. Cell Biol. 8 (7), 542 (2007).</li><li>Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+insect+cellular+immune+response.">The insect cellular immune response.</a> Insect Sci. 15 (1), 1 (2008).</li><li>Fauvarque, M. O., Williams, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+cellular+immunity:+a+story+of+migration+and+adhesion.">Drosophila cellular immunity: a story of migration and adhesion.</a> J. Cell Sci. 124 (9), 1373 (2011).</li><li>Nehme, N. T., Quintin, J., Cho, J. H., Lee, J., Lafarge, M. C., Kocks, C., Ferrandon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+roles+of+the+cellular+and+humoral+responses+in+the+Drosophila+host+defense+against+three+gram-positive+bacterial+infections.">Relative roles of the cellular and humoral responses in the Drosophila host defense against three gram-positive bacterial infections.</a> PLoS One. 6 (3), e14743 (2011).</li><li>Ulvila, J., Vanha-Aho, L. M., R&#228;met, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+phagocytosis+-+still+many+unknowns+under+the+surface.">Drosophila phagocytosis - still many unknowns under the surface.</a> APMIS. 119 (10), 651 (2011).</li><li>Moreira, C. G., Regan, J. C., Zaidman-Remy, A., Jacinto, A., Praq, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+hemocyte+migration:+an+in+vivo+assay+for+directional+cell+migration.">Drosophila hemocyte migration: an in vivo assay for directional cell migration.</a> Methods Mol. Biol. 769, 249 (2011).</li><li>Hillyer, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito+immunity.">Mosquito immunity.</a> Adv. Exp. Med. Biol. 708, 218 (2010).</li><li>Castillo, J. C., Robertson, A. E., Strand, M. 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+hemocytes+from+the+mosquitoes+Anopheles+gambiae+and+Aedes+aegypti.">Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti.</a> Insect Biochem. Mol. Biol. 36 (12), 891 (2006).</li><li>Jiang, H., Vilcinskas, A., Kanost, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity+in+lepidopteran+insects.">Immunity in lepidopteran insects.</a> Adv. Exp. Med. Biol. 708, 181 (2010).</li><li>Eleftherianos, I., Xu, M., Yadi, H., ffrench-Constant, R. H., Reynolds, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmatocyte-spreading+peptide+(PSP)+plays+a+central+role+in+insect+cellular+immune+defenses+against+bacterial+infection.">Plasmatocyte-spreading peptide (PSP) plays a central role in insect cellular immune defenses against bacterial infection.</a> J. Exp. Biol. 212 (12), 1840 (2009).</li><li>Ribeiro, C., Breh&#233;lin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+haemocytes:+what+type+of+cell+is+that.">Insect haemocytes: what type of cell is that.</a> J. Insect Physiol. 52 (5), 417 (2006).</li><li>Beetz, S., Brinkmann, M., Trenczek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+between+larval+and+pupal+hemocytes+of+the+tobacco+hornworm,+Manduca+sexta,+determined+by+monoclonal+antibodies+and+density+centrifugation.">Differences between larval and pupal hemocytes of the tobacco hornworm, Manduca sexta, determined by monoclonal antibodies and density centrifugation.</a> J. Insect Physiol. 50 (9), 805 (2004).</li><li>Beetz, S., Holthusen, T. K., Koolman, J., Trenczek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+hemocyte+counts+with+different+developmental+parameters+during+the+last+larval+instar+of+the+tobacco+hornworm,+Manduca+sexta.">Correlation of hemocyte counts with different developmental parameters during the last larval instar of the tobacco hornworm, Manduca sexta.</a> Arch. Insect Biochem. Physiol. 67 (2), 63 (2008).</li><li>Eleftherianos, I., Joyce, S., ffrench-Constant, R. H., Clarke, D. J., Reynolds, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+tri-trophic+interaction+between+insects,+nematodes+and+Photorhabdus.">Probing the tri-trophic interaction between insects, nematodes and Photorhabdus.</a> Parasitology. 137 (11), 1695 (2010).</li><li>Qayum, A. A., Telang, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Protocol+for+Collecting+and+Staining+Hemocytes+from+the+Yellow+Fever+Mosquito+Aedes+aegypti.">A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti.</a> J. Vis. Exp. (51), e2772 (2011).</li></ol>

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医学的に重要な昆虫や鱗翅目昆虫のモデルから血球を抽出するための方法は、以前に9、14が報告されている。血球抽出方法は、昆虫種によると、昆虫とその形態学的特徴の発達段階に適応している。たとえば、 マンドゥカの幼虫体液から分離は容易に腹部15の端の近くに湾曲した角を切り取ることによって行うことができる。ナメクジの幼虫（Limacodidae）この?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td>マイクロピペットプラー</td><td>サターインスツルメンツ</td><td> P-1000 </td><td></td></tr><tr><td>ホウケイ酸ガラス管</td><td>サターインスツルメンツ</td><td> B100-50から10 </td><td>外径：1.0、ID：0.50ミリメートル</td></tr><tr><td>グレース培地</td><td>シグマ</td><td> G8142 </td><td></td></tr><tr><td>ウシ胎仔血清</td><td>サーモフィッシャーサイエンティフィック</td><td> SH3007102 </td><td>不活性化された熱</td></tr><tr><td>デュモン＃5鉗子</td><td>ファイン科学ツール</td><td> 11252から40 </td><td></td></tr><tr><td>ノイバウアー血球計数器</td><td> Hausser科学</td><td> 3200 </td><td></td></tr><tr><td>プラスチックtubiNG </td><td>トライテック</td><td> TT-3-32OD </td><td>外径：3/32&#39;&#39;、番号：1/32 &#39; </td></tr><tr><td>ガラス医療シリンジ</td><td>フォルトゥナ·オプティマ</td><td> D-97877 </td><td> 50mlの体積</td></tr><tr><td>平滑末端針</td><td>小型部品</td><td> NE-162PL-25 </td><td> 16ゲージ×1 &quot;長さ</td></tr></tbody></table>

a simple protocol for extracting hemocytes from wild caterpillars

昆虫血球は（哺乳類白血球に相当）昆虫のライフサイクル全体で1いくつかの生理学的過程において重要な役割を果たしている。鱗翅目（蛾や蝶）や双翅目（ハエ真）の受注に属する昆虫の幼虫の段階では、血球はリンパ腺（専門造血器官）や胚細胞から形成され、大人の段階まで行うことができる。胚性血球細胞は、炎症時の開発および走化性調節における細胞遊走に関与している。彼らはまた、細胞のアポトーシスに参加し、胚発生に必須である2。血球は、細胞拡散、細胞凝集、結節の形成、食作用および外国の侵略者3のカプセル化などのいくつかの機能を含む昆虫自然免疫応答の細胞アームを仲介する。彼らは、広報など、また、感染時に特定の昆虫の体液性防御をオーケストレーションするための責任がある抗菌ペプチドおよび他のエフェクター分子4,5のoduction。血球形態と機能は、主にショウジョウバエ、 キイロショウジョウバエ 6,7、蚊ネッタイシマカやハマダラカ 8,9とスズメガの幼虫、 たばこスズメガ 10、11を含む、遺伝的または生理学的な昆虫モデルで研究されてきた。しかし、少しの情報は、現在野生の12から収集されたもの、特に、非モデル昆虫種の血球の多様性、分類、形態と機能について存在する。 ここでは、野生の毛虫から血球を抽出するためのシンプルで効率的なプロトコルを記述します。我々は最後から二番目の齢Lithacodes蛭 （黄色肩幅のスラグ蛾）（ 図1）とEuclea delphinii（とげのオーク·スラグ）幼虫（鱗翅目：Limacodidae）を使用し、体液の十分なボリューム（昆虫血液）を表示単離され、血球の数値は個々の幼虫からカウントすることができます。この方法は効率的にこれらの種においてだけでなく、フィールドから収穫他の関連する鱗翅目の幼虫における血球の種類を研究するために使用することも、容易に微生物や寄生生物13感染後血球機能を調査するために設計された免疫学的測定法と組み合わせることができます。

本明細書中で説明されたプロトコルは、個々の幼虫から昆虫体液の10〜20μlの最小体積の収集を可能にします。このメソッドを使用して収集血球は細胞凝集、メラニンの欠陥、組織片などの汚れから保護されています。したがって血球は容易に顕微鏡下で観察し、計数することができ、いくつかの昆虫が少ないホスール内で観察することができます。私たちは、サンプル中の血球の大部分はpla...

Watch this Scientific Journal Video about A Simple Protocol for Extracting Hemocytes from Wild Caterpillars at JoVE.com

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

昆虫血球は、昆虫開発のすべての段階を通して免疫および非免疫の両方の多くの重要な機能を、実施しています。血球の種類と機能の私達の現在の知識では、昆虫の遺伝的モデルに関する研究から得られている。ここでは、野生の毛虫から血球を定量化し、可視化、抽出するための方法を提示する。

ワイルド毛虫から血液細胞を抽出するための単純なプロトコル

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

a-simple-protocol-for-extracting-hemocytes-from-wild-caterpillars

Research

JoVE Journal

Biology

16.1K ビュー.  The George Washington University. 昆虫血球は、昆虫開発のすべての段階を通して免疫および非免疫の両方の多くの重要な機能を、実施しています。血球の種類と機能の私達の現在の知識では、昆虫の遺伝的モデルに関する研究から得られている。ここでは、野生の毛虫から血球を定量化し、可視化、抽出するための方法を提示する。

ビデオ: A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

モスキート子育てのためのプロトコル（A. gambiae）

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

生物学

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

パイロシーケンシング：正確なジェノタイピングのための簡単​​な方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4).  Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6).  For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6).  Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly.  In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons.  The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

ラット上頚神経節（SCG）から培養交感神経のためのプロトコル

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

A Protocol for the Production of KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.
Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca2+-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton. 
Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.

This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

KLRG1テトラマーの生産のためのプロトコル

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

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 ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

精巣細胞懸濁液の調製のためのシンプルで効率的な手法

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

SIVQ-LCM Protocol for the ArcturusXT Instrument

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an advanced, rapidly customizable laser dissection platform technology. In this report, we describe the integration of the image analysis software Spatially Invariant Vector Quantization (SIVQ) onto the ArcturusXT instrument. The ArcturusXT system contains both an infrared (IR) and ultraviolet (UV) laser, allowing for specific cell or large area dissections. The principal goal is to improve the speed, accuracy, and reproducibility of the laser dissection to increase sample throughput. This novel approach facilitates microdissection of both animal and human tissues in research and clinical workflows.

SIVQ-LCM is an innovative approach that harnesses a computer algorithm, Spatially Invariant Vector Quantization (SIVQ), to drive the laser capture microdissection (LCM) process. The SIVQ-LCM workflow greatly improves the speed and accuracy of microdissection, with applications in both the research and clinical settings.

ArcturusXT楽器のSIVQ-LCMプロトコル

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

血管内皮増殖因子の評価のための簡易バイオアッセイ

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

A Method for Extracting Pigments from Squid Doryteuthis pealeii

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities are supported in part by the areal expansion and retraction of pigmented organs known as chromatophores. While it is known that the chromatophores contain a tethered network of pigmented granules, their structure-function properties have not been fully detailed. We describe a method for isolating the nanostructured granules in squid Doryteuthis pealeii chromatophores and demonstrate how their associated pigments can be extracted in acidic solvents. To accomplish this, the chromatophore containing dermal layer is first manually isolated using a superficial dissection, and the pigment granules are removed using sonication, centrifugation, and washing cycles. Pigments confined within the purified granules are then extracted via acidic methanol solutions, leaving nanostructures with smaller diameters that are void of visible color. This extraction procedure produces a 58% yield of soluble pigments isolated from granules. Using this method, the composition of the chromatophore pigments can be determined and used to provide insight into the mechanism of adaptive coloration in cephalopods.

A Method for Extracting Pigments from Squid Doryteuthis pealeii

A protocol for the extraction of pigments from the nanostructured granules in squid Doryteuthis pealeii chromatophores is presented.

イカから顔料を抽出する方法<em&gt; Doryteuthis pealeii</em

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.