Name: in vivo assessment of rodent plasmodium parasitemia and merozoite invasion by flow cytometry
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Description: Watch this Scientific Journal Video about In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry at JoVE.com

私たちは、国立保健医療研究評議会（APP605524、490037と1047082を付与）、オーストラリア研究会議（DP12010061を付与）、オーストラリア国立共同研究インフラ戦略とイノベーション省から教育投資ファンド、産業界からの支援に資金を提供しアクノリッジ、科学·研究。 PMLは、オーストラリアの大学院賞を受賞です。

すべての手順は、マッコーリー大学の方針に従って行われ、国立保健医療研究評議会（NHMRC）練習のオーストラリアのコードに適合​​した。仕事はなしARAが2012/017承認されておらず、マッコーリー大学の動物倫理委員会から得た同意の倫理で行った。特に明記しない限り、全ての実験は、SJL / Jマウスで行った。 1.マウスおよび実験マラリア感染<ol><li> 12：12時間の明暗サイクルで制御された温度（21℃）下でのハウスマウス。 </li><li>凍結保存したPのアリコートを解凍5％の寄生赤血球（PRBCを）およびC57BL / 6ドナーマウスの腹腔内腔に200μlのを注入するとchabaudiアダミの DS。 </li><li>このプロトコルの4 - 第3節で説明したように、フローサイトメトリーを用いて2日 - ごとに1ドナーマウスの寄生虫血を監視します。ドナーが5に達すると - 15％の寄生虫血を次のように、心臓穿刺により血液を収集してください。 <ol><li>ヘパリン溶液を吸引し、100μlのマウスリンゲル液（MTR）で（300 U / mlのヘパリン（154ミリモルのNaCl、5.6のKCl、1mMのMgCl 2、2.2のCaCl 2、20mMのHEPES、10mMのグルコース、30 U / mlの26 G針を有する1ミリリットル注射器にヘパリン、pHが7.4、0.22μmのフィルター滅菌））。 </li><li>ペダル引っ込め反応および角膜反射の欠如によって麻酔の所望の深さを確認し、5％イソフルオラン吸入によりマウスを麻酔。 </li><li>その側にマウスを置き、垂直にちょうど肘下に、リブを通して、心臓に針を挿入します。ゆっくりとシリンジプランジャーを引き出し、0.5まで針を回転させる - 血液1mlのが得られる。 </li><li>麻酔下で頸椎脱臼により人道的安楽死を実行します。 </li></ol></li><li>犠牲にするときドナーが10％の寄生虫血であった場合（ すなわち、血液が9×10 5を含むことになる9×10 6 RBC /μLの血球数を想定したドナー血液の体積あたりのPRBCをを計算PRBC /μL）。クレブスで寄生虫を血液を希釈食塩水（126 mMの塩化ナトリウム、2.5mMのKCl、25mMのNaHCO 3水溶液、1.2ミリモルのNaH 2 PO 4、1.2のMgCl 2、2.5mMのCaCl 2、0.2％グルコース、pH7.2の、滅菌し、0.22μMフィルター）をバッファリング濃度が200μLあたり1×10 4 PRBCをされ、RBC標識および輸血実験に必要なマウスの腹腔内腔に200μlのを注入するようにします。 </li><li>このプロトコルの4 - セクション3に記載したように、フローサイトメトリーを用いて2日 - ごとに1寄生虫血を監視します。 </li></ol> RBCおよび輸血の2ラベル<ol><li> ステップ1.3で説明したように心臓穿刺によりマウスからヘパリン化血液を収集し、氷上に置きます。すべての回で、4℃で血を保管してください。注射されるマウスあたり約200μlの血液を収集する。必要に応じて、2つのサンプルに血液を分割し、必要に応じて一つのサンプルを扱う。 注：治療SAMPのこのようにして、侵略ルは、対照、未処理のサンプル（概略については図1を参照）と比較することができる。 </li><li>各蛍光RBCラベルの2倍溶液を調製する。 <ol><li>濃度が20μg/ mlのあるようにMTRにアトー633-N-ヒドロキシ（アトー633-NHS）の2mg / mlのストック溶液を希釈する。 </li><li>濃度を250μg/ mlとなるようにMTRにおけるビオチンのN-ヒドロキシスクシンイミド（NHSビオチン）の25 mg / mlのストック溶液を希釈する。 </li></ol></li><li>つのチューブに血液サンプルを分割し、一本のチューブ、および他のチューブに2×ビオチンNHSの等量に2倍アトー633-NHS溶液の等量を加える。すぐに混ぜる。一定のゆっくり混合しながら1時間4℃で血液をインキュベートするか、あるいは、すべての10のサンプルをミックス - 15分。 </li><li>次のようにMTRC（MTR + 0.5％ウシ血清アルブミン（BSA）、滅菌し、0.22μMフィルター）で標識された血液を3回洗浄する。 <ol><li> 、MTRCの少なくとも2ボリュームを追加し、5分間750×gで遠心、上清を除去し、resuspenMTRCの少なくとも2倍量のペレットはd。 2回以上繰り返します。 </li><li>最終洗浄、5分間750×gで遠心分離し、異なる組み合わせでペレット化した血液を組み合わせた後（ すなわち、未処理アトー633 /ビオチン治療、未処理ビオチン/はアトー633処置）。注射されるマウスあたり200μlの容量を最大にするMTRを追加します。 </li></ol></li><li>次のように血管内ネズミマラリアに感染したマウスまたは未感染のコントロールにラベル血液を注入： <ol><li>寄生虫の増員生殖のピーク時には15％の寄生虫血 - 2での輸血を行います。 注：これは暗サイクルを通してほぼ中間に発生（ すなわち、12〜 -リバースライトサイクルで午後2時-通常の光サイクルで午前2時、または12）。 </li><li> 5-10分間加熱ランプを使用して暖かいマウスは、血流を増加させるが、マウスを過熱しないように予防措置を取る。拘束装置にマウスを置き、血管内の血液（約1ラベル200μL注入する- 2×10 9 RBを尾静脈を介してCS）。 </li></ol></li><li>セクション3で説明したように、注射後の異なる時点で血液サンプルを収集する。 注：侵入率はレシピエントマウスの寄生虫血に応じて、注射後10分未満を定量することができる。 </li></ol>フローサイトメトリーのための血液試料と準備の3コレクション<ol><li>次のように実験の日に、染色サンプルあたりのソリューションに加え、余分な50μlのを準備します。 <ol><li>ジメチルスルホキシド中で、-20℃（DMSO）で保存6mMのJC-1のアリコートを解凍。 </li><li>ウォーム50サンプルあたりMTRCのμL、プラス37℃、余分な。 </li><li>最終濃度が12μMになるように継続的に予め温めMTRCにボルテックスしながら、JC-1を追加します。これは、JC-1の凝集体の形成を減少させるために行われる。凝集体は、フォームをすれば、このような遠心分離管は染色を防ぐことができますしないでください。 注：集計少量の結果には影響しません。 </li><li>抗追加CD45 APC eFluor 780及び抗CD71 PerCPをeFluor 710最終濃度を1μg/ mlとなるように。ラベルRBC実験を実施した場合、最終濃度が1μg/ mlのであるように、ストレプトアビジンPE-Cy7はを追加します。 </li></ol></li><li>テール内の血管の尾や裂傷の先端の切断によりテール出血を実行します。小さな量るボートやスライドガラス上にテール一滴の血液を置きます。採血後、出血が停止したことを確認してください。染色溶液50μlに尾血液のピペット3μlを37℃に予め加温し、20分間37℃でサンプルをインキュベートする。 </li><li> （355 nmのレーザーを使用する場合）を蒸留水中で-20℃で保存し、4mMのヘキスト33342または（405 nmのレーザーを使用する場合）を蒸留水中で-20℃で保存し2mMのヘキスト34580のアリコートを解凍。それぞれ、サンプルあたり500μLを準備し、プラス余分な、MTRCでヘキストの4μMまたは2μMソリューションの。 </li><li> 4μMヘキスト500μlのを追加します。サンプル33342または2μMのヘキスト34580と室温で20分間インキュベートする。 </li><li> 4℃で3分間750×gで遠心細胞、上清を捨てるには、フローサイトメトリーで700μlのMTRCを追加し、分析する。 </li></ol> 4.フローサイトメトリー<ol><li> 355/488/633 nmレーザーまたは405/488/633 nmのレーザー機器を使用してサンプルを分析する。 </li><li>分析の前にすぐに35μMのセルストレーナーを通してフィルター試料。サンプルと再使用との間に蒸留水でセルストレーナーをすすぐ。 </li><li> （ - サンプルあたり千万総イベント通常百万）最小の人口は、少なくとも500のイベントが含まれているように、十分なイベントを記録します。 </li><li> 355 nmのレーザーを使用してヘキスト33342エキサイトと50分の460フィルタを通してイベントを検出。 405 nmのレーザーを使用してヘキスト34580エキサイトと50分の460フィルタを通してイベントを検出。エキサイトJC-1、抗CD71 PerCPをeFluor 710とストレプトアビジンPE-Cy7は488 nmのレーザーを使用し、40分の530を介してイベントを検出し、40分の692、および750LP各フィルタLY。 633nmのレーザーを用いアトー633及び抗CD45 APC eFluor 780を励起し、それぞれ30分の670または750LPフィルターを通してイベントを検出。 </li><li>次のように適当なフローサイトメトリー分析ソフトウェアを用いて解析し、補正を行う。 <ol><li>全細胞を選択し、ノイズを排除し、破片、およびFSC / SSCの特性（ 図2A）に基づく血小板。いずれかのトリガーパルス幅（ 図2B）または高さの比（ 図2C）にFSCピーク面積を使用してベース単一細胞を選択する。 </li><li>成熟した赤血球を選択し、PerCPをeFluor 710とAPC eFluor 780チャンネル（ 図2D）における負の蛍光に基づいて、赤血球前駆細胞および白血球を除外する。 </li><li>ラベルRBC実験を実施する場合は、PE-Cy7はとアトー633蛍光に基づいてラベルRBCの各集団を選択し、これらを別々に（ 図3A）を分析。 </li><li> JC-1とヘキスト（両方に陽性蛍光に基づいてPRBCをを選択<strオング&gt;図2E-H）。 </li></ol></li></ol> 5.計算と統計<ol><li> RBCの総数でPRBCを数で割ることによって寄生虫血症を計算する（ すなわち、ゲートG4内のイベントの数で割ったゲートQ2のイベント数）。 2標識された集団の浸潤アッセイの寄生虫を行う場合（すなわち、集団における標識されたRBCの数により標識PRBCを数で割ることによって計算することができる、すなわち、ゲートL1及びQ2の両方にあるイベントの数、イベントの数で割ったものゲートL1で）。 NOTE：寄生虫がかなり内因性寄生虫循環メロゾイトの数などの要因に応じて変化する標識。このため、個々のマウスにおける制御標識集団の寄生虫で除算処理した標識された集団の寄生虫のように計算される寄生虫比を報告することが有用である。可能な色素の影響を補正するために、レポートの結果2染料の組み合わせ全体の平均寄生虫比だ。 </li><li>仮定の平均として1で1 標本t -testを使用して比率の統計的有意性を決定します。 </li></ol>

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著者らは、開示することは何もない。

我々は、in vivoでのサンプルの両方寄生虫およびメロゾイトの侵入を測定するための方法を記載している。寄生虫血の測定に関しては、この方法は、それによって偽陽性事象の数を減少させる、PRBCを区別することができるHJ-赤血球における以前の方法10-13に優る利点を提供する。 HJ-RBCは、ヒトでは通常まれですが、いくつかの研究では、これらの細胞とげっ歯類寄生虫の正確な...

The clinical symptoms associated with malaria occur during the Plasmodium parasite&#8217;s asexual replicative cycle within red blood cells (RBCs). Merozoites, released during the liver stage of infection, quickly attach to and invade RBCs. After gaining entry into the cell, the parasite grows and matures, eventually undergoing schizogony, splitting open the cell, and releasing a cluster of newly formed merozoites which go on to repeat this cycle. As such, an assessment of malaria infection often involves monitoring both parasitemia, which is the percentage of RBCs appropriated by one or more parasites, and the rate of merozoite invasion into uninfected RBCs. 
Flow cytometry is a powerful tool which can be used to record the properties of vast numbers of cells in a short period of time. This technique has clear applicability for the measurement of malaria parasitemia and invasion, and offers several advantages over traditional microscopy techniques. These include the accurate measurement of very low parasitemia, which would be prohibitively time consuming by microscopy, the unbiased nature of the measurement, and the ability to measure multiple cell parameters simultaneously. Flow cytometry is widely used to determine both parasitemia and merozoite invasion in in vitro culture1-9, however, techniques for measuring these parameters in vivo are less well developed, and can be complicated by the presence of additional cell types which interfere with analysis. No assays have been described for measurement of in vivo invasion, and while some assays exist for the analysis of in vivo parasitemia, these lack the ability to distinguish between parasitized RBCs (pRBCs) and RBCs containing Howell-Jolly bodies (HJ-RBCs)10-13. The later issue is particularly important as in mice HJ-RBCs may account for up to 0.9% of mature RBCs14-16, thereby preventing the accurate measurement of low parasitemia. 
We have previously demonstrated an approach for the measurement of parasitemia and merozoite invasion in a rodent model of malaria infection14. Here, we provide a more detailed protocol and accompanying video. This approach builds on previous methodologies and allows for the accurate identification of parasitized RBCs, as distinct from leukocytes, RBC progenitors, and HJ-RBCs. Additionally, this assay allows the simultaneous measurement of merozoite invasion into two labeled RBC populations, a treated, or target, population, and a control population, thereby providing a robust platform for the assessment of invasion into different cell types.

<table><tbody><tr><td>bisBenzimide H 33342 trihydrochloride</td><td>Sigma-Aldrich</td><td>B2261</td><td>Hoechst 33342. Store a 4 mM stock solution at -20 &amp;deg;C in distilled water</td></tr><tr><td>Hoechst 34580</td><td>Sigma-Aldrich</td><td>63493</td><td>Store a 2 mM stock solution at -20 &amp;deg;C in distilled water</td></tr><tr><td>JC-1 Dye</td><td>Life Technologies</td><td>T-3168</td><td>Store small aliquots of 6 mM stock solution at -20 &amp;deg;C in DMSO</td></tr><tr><td>Anti-Mouse CD45 APC-eFluor 780</td><td>eBioscience</td><td>47-0451-80</td><td>Clone 30-F11</td></tr><tr><td>Anti-Mouse CD71 PerCP-eFluor 710</td><td>eBioscience</td><td>46-0711-80</td><td>Clone R17217</td></tr><tr><td>Atto 633 NHS ester</td><td>Sigma-Aldrich</td><td>1464</td><td>Atto 633-NHS. Store a 2 mg/ml stock solution at -20 &amp;deg;C in DMF</td></tr><tr><td>EZ-Link Sulfo-NHS-LC-Biotin</td><td>Thermo Fisher Scientific</td><td>21335</td><td>Biotin-NHS. Store a 25 mg/ml stock solution at -20 &amp;deg;C in DMF</td></tr><tr><td>Streptavidin PE-Cyanine7</td><td>eBioscience</td><td>25-4317-82</td><td>Streptavidin PE-Cy7</td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H478</td><td></td></tr><tr><td>35 &amp;micro;M filter cap tubes</td><td>Becton Dickinson</td><td>352235</td><td></td></tr><tr><td>Flow cytometer: BD LSRFortessa</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Flow cytometer: BD FACSAria II</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Flow cytometer: BD Influx</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Flow cytometer: CyAn ADP Analyzer</td><td>Beckman Coulter</td><td></td><td></td></tr></tbody></table>

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.

寄生虫の測定。 寄生虫血の測定のために、血液細胞が最初に選択されるべきであり、ノイズ、破片および血小板は、FSC / SSCの特性（ 図2A）に基づいて、除外した。使用するフローサイトメーターに応じて、単一の細胞を、次いで、面積比（ 図2C）のいずれかでトリガーパルス幅（ 図2B）、またはFSCピーク高さに基づいて選択されるべ?...

Watch this Scientific Journal Video about In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry at JoVE.com

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

マラリア原虫が侵入し、赤血球内で複製する。メロゾイトの侵入および寄生虫の正確な評価は、マラリア感染の経過を評価するので、非常に重要です。ここでは、マラリアのマウスモデルにおいて、これらのパラメータを測定するためのフローサイトメトリーベースのプロトコルを説明する。

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 ...

in-vivo-assessment-rodent-plasmodium-parasitemia-merozoite-invasion

Research

JoVE Journal

Immunology and Infection

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

9.9K ビュー.  Macquarie University. マラリア原虫が侵入し、赤血球内で複製する。メロゾイトの侵入および寄生虫の正確な評価は、マラリア感染の経過を評価するので、非常に重要です。ここでは、マラリアのマウスモデルにおいて、これらのパラメータを測定するためのフローサイトメトリーベースのプロトコルを説明する。 

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

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

げっ歯類のマラリア原虫の遺伝的交雑の生産のためのプロトコル

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

免疫・感染学

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

クローズ頭蓋窓を使用してマウス脳の微小循環の生体顕微鏡

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

神経科学

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

脾臓の生体顕微鏡検査：寄生虫モビリティと血流​​の定量的解析

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

脳から隔離された白血球の単離と解析<em&gt;マラリアベルゲイ</em&gt; ANKA感染マウス

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

高収率の精製<em&gt;熱帯熱マラリア原虫</emオプソニン抗体アッセイにおける使用のための&gt;メロゾイト

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

アン<em&gt;インビトロ</em&gt; HIV重感染のコンテキストでのマラリアに対する免疫応答を測定するためのモデル

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

げっ歯類マラリア寄生虫の現象分析無性および性的血液段階と蚊の段階

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

磁気共鳴映像法を用いた実験的大脳マラリアモデルにおける浮腫発生と微小血管病変のin vivo追跡

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

医学

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 ...

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective immunity to malaria is still poorly understood, mainly due to methodological limitations of long-term Plasmodium culture, especially for Plasmodium vivax. Most studies have focused on adaptive immunity protection against malaria by antibodies, which play a key role in controlling malaria. However, the sterile protection induced by attenuated Plasmodium sporozoites vaccines is related to cellular response, mainly to cytotoxic T lymphocytes, such as CD8+ and gamma delta T cells (&#947;&#948; T). Hence, new methodologies must be developed to better comprehend the functions of the cellular immune response and thus support future therapy and vaccine development. To find a new strategy to analyze this cell-mediated immunity to Plasmodium blood-stage infection, our group established an in vitro assay that measures infected red blood cell (iRBC) killing by cytotoxic lymphocytes. This assay can be used to study cellular immune response mechanisms against different Plasmodium spp. in the blood stage. Innate and adaptative cytotoxic immune cells can directly eliminate iRBCs and the intracellular parasite in an effector:target mechanism. Target iRBCs are labeled to evaluate cell viability, and cocultured with effector cells (CD8+ T, &#947;&#948; T, NK cells, etc.). The lysis percentage is calculated based on tested conditions, compared to a spontaneous lysis control in a flow cytometry-based assay. Ultimately, this killing assay methodology is a major advance in understanding cell-mediated immunity to blood-stage malaria, helping uncover new potential therapeutic targets and accelerate the development of malaria vaccines.

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Here we describe a new method to help elucidate the mechanisms of cellular immunity to Plasmodium during the blood stage of infection. This is an in vitro assay that measures infected red blood cell killing by cytotoxic lymphocytes.&#160;

インビトロ 細胞傷害性リンパ球による マラリア原虫感染赤血球殺傷のアッセイ

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective ...

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

要約

さらに動画を探す

この動画の章

げっ歯類のマラリア原虫の遺伝的交雑の生産のためのプロトコル

クローズ頭蓋窓を使用してマウス脳の微小循環の生体顕微鏡

脾臓の生体顕微鏡検査：寄生虫モビリティと血流の定量的解析

脳から隔離された白血球の単離と解析

高収率の精製

アン

げっ歯類マラリア寄生虫の現象分析無性および性的血液段階と蚊の段階

磁気共鳴映像法を用いた実験的大脳マラリアモデルにおける浮腫発生と微小血管病変のin vivo追跡

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

インビトロ細胞傷害性リンパ球によるマラリア原虫感染赤血球殺傷のアッセイ

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

要約

さらに動画を探す

この動画の章

げっ歯類のマラリア原虫の遺伝的交雑の生産のためのプロトコル

クローズ頭蓋窓を使用してマウス脳の微小循環の生体顕微鏡

脾臓の生体顕微鏡検査：寄生虫モビリティと血流​​の定量的解析

脳から隔離された白血球の単離と解析

高収率の精製

アン

げっ歯類マラリア寄生虫の現象分析無性および性的血液段階と蚊の段階

磁気共鳴映像法を用いた実験的大脳マラリアモデルにおける浮腫発生と微小血管病変のin vivo追跡

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