私たちは、トリパノソーマ、生物学上の一般的なアドバイスのためのジョージ・クロスを承認したいと思います。この作品はまた、DS、MRMへのNSF大学院研究フェローシップ（DGE-1325261）及びFNPにNIH / NIAID（助成金＃のAI085973）にビル・アンド・メリンダ・ゲイツ財団GCEの助成金によってサポートされていました。我々は、I-SCEI遺伝子と認識部位を含む菌株の使用をガラドリエルあばら家-マイナーに感謝します。

注：手順を通して、それを氷上で細胞を維持することが必要です。コールドメディアも全体で使用されるべきです。 1.サンプル収穫<ol><li> 100万/ mlの - 0.5の密度にリスター427株血流のトリパノソーマを成長させます。これは、寄生虫数の少ない培養を開始するのが最善の方法です。 1500×gで10分間、50×10 6細胞/サンプルをスピンダウン。正と負の抗体コントロールとして後で使用するために文化の中で1×10 6個の細胞を残すようにしてください。 注：このプロトコルは、（詳細説明を参照）、動物の血液から単離されたトリパノソーマに使用することができます。 </li><li>ピペットまたは上清の大部分を捨てる（約750μLを残します）。 1.5ミリリットルマイクロ遠心チューブに細胞を移します。 </li><li>微量で5200×gで4分間、4℃で細胞をスピンし、上清を除去します。 </li></ol> 2.磁気標識<ol><li> 150μlの培養培地中で再懸濁細胞（HMI-9のwi適切な希釈で例えば番目血清）+一次抗VSG抗体。 注：抗VSG抗体は、家の中で作られ、1時50分の希釈で使用されています。フルオロフォアで標識されていない一次抗VSGの抗体を使用することが重要です。 </li><li> 10分間、4℃の低温室に8 - スピード6で渦アダプタ-60を使用して、渦細胞。血清で千μlの冷間HMI-9から800で洗います。 </li><li> 5200×gで4分間、4℃でスピンし、上清を吸引除去します。血清を1ミリリットルHMI-9で洗浄し、上記のようにスピン。上清を吸引除去します。血清とHMI-9の100μlの再懸濁ペレット。 </li><li> （MSC）マイクロビーズをソートする磁気活性化細胞の110μLを加えます。一次抗VSG抗体に応じて抗マウス、抗ウサギ、または抗ビオチンビーズを使用してください。よく混ぜます。 10分間、4℃の低温室での渦細胞。 </li><li>細胞はボルテックスされているが、分離磁石に1磁気活性化分離カラム/サンプルを設定します。再を持っているようにしてくださいフロースルーを収集する列の下ceptacle（私たちは15ミリリットルの遠心分離管を使用します）。低温室で装置を設定します。 </li><li>プライムカラムに血清を使用して、HMI-9の2ミリリットルを追加します。フロースルー各試料から収集する列の下に15ミリリットルの遠心分離管場所をプライミングした後。 </li><li>ステップ2.4で10分間インキュベーションした後、血清をコールドHMI-9 800〜μlを添加します。 5200×gで4分間、4℃でスピンし、上清を除去し、未結合抗体を除去します。血清およびリピートは100μlの上清を残して、HMI-9の1ミリリットルで洗浄します。 </li><li>激しく視覚的に遠心管をフリックペレットを完全に再懸濁されていることを確認するために、チューブを点検します。細胞が十分に再懸濁されていることを確認するには、上下の血清およびピペットでHMI-9の1ミリリットルを追加します。 </li></ol> 3.磁気分離<ol><li>上記のように列がプライミングされていることを確認します。カラムに細胞を適用します。フロースルー非支配VSを発現する細胞とを収集カラムを通って流れるように7分 - G. 1 mlの培地+トリパノソーマは、一般的に6を取ります。 </li><li>ウォッシュ血清とHMI-9の1ミリリットルで2回およびフロースルー同じチューブのステップ3.1のように集めます。 </li><li>除算フロースルー（3ミリリットル）2 1.5ミリリットルのマイクロチューブに。 注：これらは、後に組み合わせることができるか、半分など 、RNAを単離することができます</li><li>磁石から列を削除します。カラムに血清を使用して、HMI-9の3ミリリットルを追加し、開始、支配的なVSGを発現する細胞を得るために15ミリリットルの遠心分離管に列を急落。後の分析のために溶出した物質の300μLを削除します。氷の上でこれらの細胞を保管してください。 </li><li>陽性および陰性対照として使用するために、37℃で増殖対照細胞の出発培養物からの約100万個の細胞を取り1.5ml微量チューブに、それらをピペット（それらは頻繁に切り替えることが予想されていません）。 注：ポジティブコントロール細胞はステップ4.1でフルオロフォア標識抗体で染色されます。ネガティブコントロール細胞REMAプロトコルの残りの部分全体に染色されていないインチ</li><li>スピンフロースルーサンプルおよび抗体陽性対照試料4℃で4分間、5200×gで、上清を除去します。 注：溶出セルの別の300μlのアリコートを、抗VSG染色の陽性対照として使用することができます。 </li></ol> 4.染色は、汚染物質やスイッチャーを識別するために、 <ol><li>再懸濁し、フロースルーサンプルおよび100μlのHMI-9適切な希釈で血清+一次、フルオロフォア標識、抗VSG抗体とにおけるポジティブコントロール細胞。 注：標識抗VSG抗体は家で作られ、1の希釈で使用されている：1,000。フルオロフォア標識、抗VSGはstep.Thereは、ステップ3.5以下の二つのマイクロ遠心チューブ内のフロースルーサンプルの3ミリリットルであるべきMACS分離で非標識抗体と同じソースからすることができます。 </li><li>フローサイトメトリーによってフロースルーのすべてを分析するために、一方の再SUS 2マイクロ遠心チューブ内でペレットを結合血清+抗VSG抗体を使用して、HMI-9の100μl中のサンプルを保留中。よく混ぜます。 </li><li> 10分間、4℃の低温室での渦細胞。千μlの冷間HMI-9血清との - 800を追加します。スピンサンプル5200×gで4分間、4℃で、非結合抗体を除去するために、上清を除去します。 </li><li>血清とHMI-9の1ミリリットルで細胞を洗浄。スピンサンプル5200×gで4分間、4℃で、上清を除去します。この最後のスピンの間に、ステップ3.4および陰性対照試料から溶出し、サンプルをスピンダウン。 </li><li>血清で148.5μlのHMI-9、25μlの絶対計数ビーズと1.5μlのヨウ化プロピジウム染色溶液（175μlの合計）：内のすべてのフロースルーと溶出サンプルを再懸濁します。 175μlの正および負の抗体対照試料を再懸濁HMI-9の血清で。絶対計数ビーズ/使用される特定のロットの25μlの数を記録してください。 </li><li>フローサイトメーターを介してすべてのサンプルを実行し、データを収集します。死細胞は、PIとして染色します<sup&gt; +。 VSGの+ - PIなどの汚染物質の染色。 VSG - -スイッチャーは、PIとして染色8。 </li><li>フローサイトメトリーデータを使用してスイッチング周波数計算します。 注：スイッチング周波数を計算する方法については、補足ファイルを参照してください<a href="https://www.jove.com/files/ftp_upload/54715/Flow_Cytometry_Calculations.docx" target="_blank">フローサイトメトリーCalculations.docx</a>および表1。 </li></ol>

<ol><li>Horn, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antigenic+variation+in+African+trypanosomes%5BTitle%5D)+AND+%22Molecular+%26+Biochemical+Parasitology%22%5BJournal%5D)">Antigenic variation in African trypanosomes.</a> Molecular & Biochemical Parasitology. 195 (2), 123-129 (2014).</li>
<li>Lamont, G. S., Tucker, R. S., Cross, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+antigen+switching+rates+in+Trypanosoma+brucei%5BTitle%5D)+AND+%22Parasitology%22%5BJournal%5D)">Analysis of antigen switching rates in Trypanosoma brucei.</a> Parasitology. 92 (Pt 2), 355-367 (1986).</li>
<li>Rudenko, G., McCulloch, R., Dirks-Mulder, A., Borst, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Telomere+exchange+can+be+an+important+mechanism+of+variant+surface+glycoprotein+gene+switching+in+Trypanosoma+brucei%5BTitle%5D)+AND+%22Mol+Biochem+Parasitol%22%5BJournal%5D)">Telomere exchange can be an important mechanism of variant surface glycoprotein gene switching in Trypanosoma brucei.</a> Mol Biochem Parasitol. 80 (1), 65-75 (1996).</li>
<li>Turner, C. M., Barry, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+frequency+of+antigenic+variation+in+Trypanosoma+brucei+rhodesiense+infections%5BTitle%5D)+AND+%22Parasitology%22%5BJournal%5D)">High frequency of antigenic variation in Trypanosoma brucei rhodesiense infections.</a> Parasitology. 99 (Pt 1), 67-75 (1989).</li>
<li>Miller, E. N., Turner, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+antigenic+types+appearing+in+first+relapse+populations+of+clones+of+Trypanosoma+brucei%5BTitle%5D)+AND+%22Parasitology%22%5BJournal%5D)">Analysis of antigenic types appearing in first relapse populations of clones of Trypanosoma brucei.</a> Parasitology. 82 (1), 63-80 (1981).</li>
<li>Horn, D., Cross, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+Trypanosoma+brucei+vsg+expression+site+switching+in+vitro%5BTitle%5D)+AND+%22Mol+Biochem+Parasitol%22%5BJournal%5D)">Analysis of Trypanosoma brucei vsg expression site switching in vitro.</a> Mol Biochem Parasitol. 84 (2), 189-201 (1997).</li>
<li>Figueiredo, L. M., Janzen, C. J., Cross, G. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+histone+methyltransferase+modulates+antigenic+variation+in+African+trypanosomes%5BTitle%5D)+AND+%22PLoS+Biol%22%5BJournal%5D)">A histone methyltransferase modulates antigenic variation in African trypanosomes.</a> PLoS Biol. 6 (7), e161(2008).</li>
<li>Boothroyd, C. E., Dreesen, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+yeast-endonuclease-generated+DNA+break+induces+antigenic+switching+in+Trypanosoma+brucei%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A yeast-endonuclease-generated DNA break induces antigenic switching in Trypanosoma brucei.</a> Nature. 459 (7244), 278-281 (2009).</li>
<li>Engstler, M., Pfohl, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hydrodynamic+Flow-Mediated+Protein+Sorting+on+the+Cell+Surface+of+Trypanosomes%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Hydrodynamic Flow-Mediated Protein Sorting on the Cell Surface of Trypanosomes.</a> Cell. 131 (3), 505-515 (2007).</li>
<li>Mugnier, M. R., Cross, G. A. M., Papavasiliou, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+in+vivo+dynamics+of+antigenic+variation+in+Trypanosoma+brucei%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The in vivo dynamics of antigenic variation in Trypanosoma brucei.</a> Science. 347 (6229), 1470-1473 (2015).</li>
</ol>

The authors have nothing to disclose.

実験技術に関しては、プロトコルの最も重要なコンポーネントは、冷たいすべてのサンプルを保っています。トリパノソーマは非常に迅速に、それらの表面9に結合した抗体の内在化が、このプロセスは、運動性に依存し、細胞を4℃に維持される限り、アッセイに影響を及ぼしません。全てのサンプルは、常に氷上で保存されるべきで、ピペッティングを25℃の実験室環境への暴露を?...

寄生原虫、 トリパノソーマは 、サハラ以南のアフリカ全域（ トリパノソーマブルセイを介して）や動物（ トリパノソーマガンビアとトリパノソーマローデシア経由）人間に影響を与える疾患の原因物質です。なお、ツェツェバエベクターの唾液を介して哺乳動物宿主に伝達されます。人間と動物、アフリカトリパノソーマ症の両方が流行地域に深刻な経済的負担を引き起こす、といくつかの薬が利用可能であるか、またはいずれかの疾患を治療するために開発中です。免疫回避のメカニズムを理解することは、トリパノソーマ症用薬の開発のために重要です。寄生虫をカバーして密な、バリアント表面糖タンパク質（VSG）コートの抗原性の変化は、主要な手段の一つであることでT.トリパノソーマは、哺乳類の抗体応答を逃れます。 VSG遺伝子の約2,000の変異体は、トリパノソーマのゲノムに存在するが、一つだけは〜15 EXPRESの1から任意の時点で転写されますシオンサイト。表現VSGの「切り替えは、「アクティブ発現部位にVSG遺伝子をコピーすることによって、または（1件）以前にサイレント発現部位の転写活性化のいずれかによって発生する可能性があります。 多くの仕事は、Tにおける抗原変異を理解することに専念してきたがトリパノソーマは 、スイッチング周波数の影響を与える要因は、まだよく理解されていません。これまでの研究では、約10 6セル2 1の順に、スイッチングがインビトロで確率的に発生している間、スイッチング周波数が非常に低いという事実によって妨げられてきました。これは、それが困難なスイッチングが最初の場所で検出が困難であるため、スイッチング周波数を減少させ、所定の係数が大きくなるかどうかを測定することができます。 2009年以前には、所定の集団でスイッチャーを単離するための方法は、長く、労働集約的でした。これらには、が支配的VSGに対して免疫化したマウスを通じてトリパノソーマを継代した後、細胞を回収日後3、または免疫蛍光4,5によって細胞の数百を数えます。別の戦略は、スイッチャー6を単離するために、薬剤耐性の選択に依存しています。 インビトロで増殖アフリカトリパノソーマは、典型的には、1つの主要な変異体、および代替変異体を発現スイッチャのはるかに小さい人口を表現する大集団で構成されているので、我々は支配的な、出発VSGとしてこの論文を通じて、主要なバリアントを参照してください。そうすることで、私たちは決してこの主要な変異体は、集団内の他の変異体よりも大きな適応度を持っていることを暗示したいです。 4時間 - ここでは、確実に3に与えられた集団における非支配的なVSGを発現しているトリパノソーマの数を測定することができる方法について説明します。この方法は、1つの与えられた遺伝子操作または薬物治療が集団に切り替え細胞の数を増加させるかどうかを確認したい場合に特に有用です。代わりにDOを発現する細胞の集団をなくししますminantは、薬を介して、または免疫学的手段によってVSGを開始し、これらの細胞を最初に支配VSGに対する抗体に結合された磁気ビーズでそれらをコーティングした後、磁気カラム上でそれらを分離することによって除去されます。切り替え集団は、フロースルーに回収し、汚染物質を識別するためのフルオロフォア標識した抗VSG抗体で再度染色します。定量化は、細胞に対するビーズの比を決定し、集団7にスイッチャの数を定量するために使用することができるように、各サンプルの絶対計数ビーズの定義された数を加算することによって達成されます。

<table><tbody><tr><td>unlabeled anti-VSG antibody</td><td>n/a</td><td>n/a</td><td>VSG antibody is made in house</td></tr><tr><td>fluorophore labeled anti-VSG antibody</td><td>n/a</td><td>n/a</td><td>VSG antibody is made in house</td></tr><tr><td>HMI-9 media</td><td>n/a</td><td>n/a</td><td>HMI-9 is made in house</td></tr><tr><td>Propidium Iodide</td><td>BD Pharmingen</td><td>556463</td><td></td></tr><tr><td>CountBright absolute counting beads</td><td>Thermofisher Scientific</td><td>C36950</td><td></td></tr><tr><td>LD columns</td><td>Miltenyi Biotech</td><td>130-042-901</td><td></td></tr><tr><td>MACS magnet</td><td>Miltenyi Biotech</td><td>130-042-303</td><td></td></tr><tr><td>MACS magnetic separator</td><td>Miltenyi Biotech</td><td>130-042-302</td><td></td></tr><tr><td>vortex adapter-60</td><td>ThermoFisher scientific</td><td>AM10014</td><td></td></tr><tr><td>flow cytometer</td><td>Coulter</td><td>n/a</td><td></td></tr><tr><td>flow cytometer analysis software</td><td>FloJo</td><td>n/a</td><td></td></tr></tbody></table>

detection of trypanosoma brucei variant surface glycoprotein switching by magnetic activated cell sorting and flow cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

ここで説明する方法は、VSG発現部位内の二本鎖切断は、人口8にスイッチャーの数を増加させたことを実証しました。ここでは、同様に発現部位で二本鎖切断を生成するように誘導されているトリパノソーマの集団からの代表的結果を示します。私たちは、二本鎖切断を生成するように誘導されていないものにこれらのトリパノソーマを比較する。 図1は...

Watch this Scientific Journal Video about Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry at JoVE.com

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

の検出<em&gt;トリパノソーマ</em磁気活性化細胞選別し、フローサイトメトリーによって&gt;バリアント表面糖タンパク質の切り替え

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

detection-trypanosoma-brucei-variant-surface-glycoprotein-switching

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JoVE Journal

Immunology and Infection

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

8.7K ビュー.  Rockefeller University. African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

ビデオ: Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer1-7. In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens7. It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to na&iuml;ve counterparts7-10. However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression11,12. Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo.
Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to na&iuml;ve MDSC 13. MDSC percentages are significantly less in lymphoid compartments of na&iuml;ve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1+ leukocytes from na&iuml;ve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1+ leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1+ leukocytes from spleens of na&iuml;ve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas na&iuml;ve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from na&iuml;ve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1+ leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1+ enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (na&iuml;ve vs. tumor- bearing) in in vivo and in vitro assays.

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

This is a rapid and comprehensive method of immunophenotyping Myeloid Derived Suppressor Cells (MDSC) and enriching Gr-1+ leukocytes from mouse spleens. This method uses flow cytometry and AutoMACS Cell Sorting to enrich for viable Gr-1+ leukocytes prior to FACS sorting of MDSC for use in vivo and in vitro assays.

フローサイトメトリーおよび自動化された磁気活性化細胞選別（AutoMACS）を使用して、ナイーブ膵腫瘍を有するマウスからミエロイド系のサプレッサー細胞（MDSC）の調製

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

医学

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

アフリカトリパノソーマにグライコダイナミクスを監視する蛍光タンパク質を使用した

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

免疫・感染学

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of malaria.

フローサイトメトリーによるげっ歯類マラリア原虫のin vivo評価とメロゾイト侵略で

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

磁気変調バイオセンシングシステムを用いた生物学的アッセイの迅速な均質な検出

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

生物学

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

ハイスループット遺伝子タギングで<em&gt;トリパノソーマ</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

ビリオンの抗原性スペクトルと細胞外ベシクルを分析するためのフローVirometry

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

表現型および機能解析のためのレア抗原特異的B細胞の検出および濃縮

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns)

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is the best method for diagnosis of first stage HAT and furthermore this parasite purification method permits serological and research investigations.
HAT is caused by Tsetse fly transmitted Trypanosoma brucei gambiense and T. b. rhodesiense. Related trypanosomes are the causative agents of animal trypanosomiasis. Trypanosome detection is essential for HAT diagnosis, treatment and follow-up. The technique described here is the most sensitive parasite detection technique, adapted to field conditions for the diagnosis of T. b. gambiense HAT and can be completed within one hour. Blood is layered onto an anion-exchanger column (DEAE cellulose) previously adjusted to pH 8, and elution buffer is added. Highly negatively charged blood cells are adsorbed onto the column whereas the less negatively charged trypanosomes pass through. Collected trypanosomes are pelleted by centrifugation and observed by microscopy. Moreover, parasites are prepared without cellular damage whilst maintaining their infectivity.
Purified trypanosomes are required for immunological testing; they are used in the trypanolysis assay, the gold standard in HAT serology. Stained parasites are utilized in the card agglutination test (CATT) for field serology. Antigens from purified trypanosomes, such as variant surface glycoprotein, exoantigens, are also used in various immunoassays. The procedure described here is designed for African trypanosomes; consequently, chromatography conditions have to be adapted to each trypanosome strain, and more generally, to the blood of each species of host mammal.
These fascinating pathogens are easily purified and available to use in biochemical, molecular and cell biology studies including co-culture with host cells to investigate host-parasite relationships at the level of membrane receptors, signaling, and gene expression; drug testing in vitro; investigation of gene deletion, mutation, or overexpression on metabolic processes, cytoskeletal biogenesis and parasite survival.

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers Diethylaminoethyl-cellulose Columns

This method of trypanosome separation from blood depends on their surface charge being less negative than mammalian blood cells. Infected blood is placed and treated on an anion-exchanger column. This method, the most fitting diagnostic for African trypanosomiasis, provides purified parasites for immunological, biological, biochemical, pharmaceutical and molecular biology investigations.

陰イオン交換器 (ジエチルアミノエチル セルロース列) による血中からのアフリカを含む細胞外の Trypanosomes の精製

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is ...

Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

Fine Needle Aspiration (FNA) is a routine diagnostic procedure essential to both medical and veterinary practices. It consists of the percutaneous aspiration of cells and/or microorganisms from palpable masses, organs or effusions (fluid accumulation in a body cavity) using a thin needle similar to the regular needle used for the venous puncture. The material collected by FNA is in general highly cellular, and the retrieved aspirate is then smeared, air dried, wet-fixed, stained and observed under a microscope. In the clinical context, FNA is an important diagnostic tool that serves as a guide to the appropriate therapeutic management. Because it is simple, fast, minimally invasive and requires limited investment in the laboratory and human resources, it is extensively used by veterinary practitioners, mostly in domestic, but also in farm animals. In studies using animal models, FNA has the advantage that it can be performed repeatedly in the same animal, enabling longitudinal studies through the collection of cells from tumors and organs/tissues over the course of the disease. In addition to routine microscopy, retrieved material can also be used for immunocytochemistry, electron microscopy, biochemical analysis, flow cytometry, molecular biology or in vitro assays. FNA has been used to identify the protozoan parasite Trypanosoma brucei in the gonads of infected mice, opening the possibility for a future diagnosis in cattle.

Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

Fine Needle Aspiration is a technique, whereby cells are obtained from a lesion or organ using a thin needle. Aspirated material is smeared, stained and examined under a microscope for diagnosis or used for molecular biology, cytometry or in vitro analysis. It is cheap, simple, quick and causes minimal trauma.

細針吸引による血管外トリパノノーマ寄生虫の検出

Fine Needle Aspiration (FNA) is a routine diagnostic procedure essential to both medical and veterinary practices. It consists of the percutaneous ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

フローサイトメトリーベースアッセイによる熱帯 熱マラリア原 虫に対する自然獲得食細胞症誘導抗体の測定

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

の検出

要約

さらに動画を探す

フローサイトメトリーおよび自動化された磁気活性化細胞選別（AutoMACS）を使用して、ナイーブ膵腫瘍を有するマウスからミエロイド系のサプレッサー細胞（MDSC）の調製

アフリカトリパノソーマにグライコダイナミクスを監視する蛍光タンパク質を使用した

フローサイトメトリーによるげっ歯類マラリア原虫のin vivo評価とメロゾイト侵略で

磁気変調バイオセンシングシステムを用いた生物学的アッセイの迅速な均質な検出

ハイスループット遺伝子タギングで

ビリオンの抗原性スペクトルと細胞外ベシクルを分析するためのフローVirometry

表現型および機能解析のためのレア抗原特異的B細胞の検出および濃縮

陰イオン交換器 (ジエチルアミノエチルセルロース列) による血中からのアフリカを含む細胞外の Trypanosomes の精製

細針吸引による血管外トリパノノーマ寄生虫の検出

フローサイトメトリーベースアッセイによる熱帯熱マラリア原虫に対する自然獲得食細胞症誘導抗体の測定

の検出

要約

さらに動画を探す

フローサイトメトリーおよび自動化された磁気活性化細胞選別（AutoMACS）を使用して、ナイーブ膵腫瘍を有するマウスからミエロイド系のサプレッサー細胞（MDSC）の調製

アフリカトリパノソーマにグライコダイナミクスを監視する蛍光タンパク質を使用した

フローサイトメトリーによるげっ歯類マラリア原虫のin vivo評価とメロゾイト侵略で

磁気変調バイオセンシングシステムを用いた生物学的アッセイの迅速な均質な検出

ハイスループット遺伝子タギングで

ビリオンの抗原性スペクトルと細胞外ベシクルを分析するためのフローVirometry

表現型および機能解析のためのレア抗原特異的B細胞の検出および濃縮

陰イオン交換器 (ジエチルアミノエチル セルロース列) による血中からのアフリカを含む細胞外の Trypanosomes の精製

細針吸引による血管外トリパノノーマ寄生虫の検出

フローサイトメトリーベースアッセイによる熱帯 熱マラリア原 虫に対する自然獲得食細胞症誘導抗体の測定

陰イオン交換器 (ジエチルアミノエチルセルロース列) による血中からのアフリカを含む細胞外の Trypanosomes の精製

フローサイトメトリーベースアッセイによる熱帯熱マラリア原虫に対する自然獲得食細胞症誘導抗体の測定