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>

<ol>
	<li>Jacobberger, J. W., Horan, P. K., Hare, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+malaria+parasite-infected+blood+by+flow+cytometry.">Analysis of malaria parasite-infected blood by flow cytometry.</a> Cytometry. 4 (3), 228-237 (1983).</li><li>Bianco, A. E., Battye, F. L., Brown, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum:+rapid+quantification+of+parasitemia+in+fixed+malaria+cultures+by+flow+cytometry.">Plasmodium falciparum: rapid quantification of parasitemia in fixed malaria cultures by flow cytometry.</a> Exp Parasitol. 62 (2), 275-282 (1986).</li><li>Makler, M. T., Lee, L. G., Recktenwald, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiazole+orange:+a+new+dye+for+Plasmodium+species+analysis.">Thiazole orange: a new dye for Plasmodium species analysis.</a> Cytometry. 8 (6), 568-570 (1987).</li><li>Heyde, H. C., Elloso, M. M., van de Waa, J., Schell, K., Weidanz, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+hydroethidine+and+flow+cytometry+to+assess+the+effects+of+leukocytes+on+the+malarial+parasite+Plasmodium+falciparum.">Use of hydroethidine and flow cytometry to assess the effects of leukocytes on the malarial parasite Plasmodium falciparum.</a> Clin Diagn Lab Immunol. 2 (4), 417-425 (1995).</li><li>Pattanapanyasat, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+malaria+parasites+in+two+different+red+blood+cell+populations+using+biotin+and+flow+cytometry.">Culture of malaria parasites in two different red blood cell populations using biotin and flow cytometry.</a> Cytometry. 25 (3), 287-294 (1996).</li><li>Grimberg, B. T., Erickson, J. J., Sramkoski, R. M., Jacobberger, J. W., Zimmerman, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+Plasmodium+falciparum+growth+and+development+by+UV+flow+cytometry+using+an+optimized+Hoechst-thiazole+orange+staining+strategy.">Monitoring Plasmodium falciparum growth and development by UV flow cytometry using an optimized Hoechst-thiazole orange staining strategy.</a> Cytometry A. 73 (6), 546-554 (2008).</li><li>Theron, M., Hesketh, R. L., Subramanian, S., Rayner, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+adaptable+two-color+flow+cytometric+assay+to+quantitate+the+invasion+of+erythrocytes+by+Plasmodium+falciparum+parasites.">An adaptable two-color flow cytometric assay to quantitate the invasion of erythrocytes by Plasmodium falciparum parasites.</a> Cytometry A. 77 (11), 1067-1074 (2010).</li><li>Bei, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flow+cytometry-based+assay+for+measuring+invasion+of+red+blood+cells+by+Plasmodium+falciparum.">A flow cytometry-based assay for measuring invasion of red blood cells by Plasmodium falciparum.</a> Am J Hematol. 85 (4), 234-237 (2010).</li><li>Clark, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RBC+barcoding+allows+for+the+study+of+erythrocyte+population+dynamics+and+P.+falciparum+merozoite+invasion.">RBC barcoding allows for the study of erythrocyte population dynamics and P. falciparum merozoite invasion.</a> PLoS One. 9 (7), e101041 (2014).</li><li>Malleret, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+robust+tri-color+flow+cytometry+assay+for+monitoring+malaria+parasite+development.">A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development.</a> Sci Rep. 1 (118), (2011).</li><li>Jimenez-Diaz, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+Plasmodium-infected+erythrocytes+in+murine+models+of+malaria+by+flow+cytometry+using+bidimensional+assessment+of+SYTO-16+fluorescence.">Quantitative measurement of Plasmodium-infected erythrocytes in murine models of malaria by flow cytometry using bidimensional assessment of SYTO-16 fluorescence.</a> Cytometry A. 75 (3), 225-235 (2009).</li><li>Jimenez-Diaz, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+detection+specificity+of+Plasmodium-infected+murine+erythrocytes+by+flow+cytometry+using+autofluorescence+and+YOYO-1.">Improvement of detection specificity of Plasmodium-infected murine erythrocytes by flow cytometry using autofluorescence and YOYO-1.</a> Cytometry A. 67 (1), 27-36 (2005).</li><li>Jun, G., Lee, J. S., Jung, Y. J., Park, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+determination+of+Plasmodium+parasitemia+by+flow+cytometry+and+microscopy.">Quantitative determination of Plasmodium parasitemia by flow cytometry and microscopy.</a> J Korean Med Sci. 27 (10), 1137-1142 (2012).</li><li>Lelliott, P. M., Lampkin, S., McMorran, B. J., Foote, S. J., Burgio, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flow+cytometric+assay+to+quantify+invasion+of+red+blood+cells+by+rodent+Plasmodium+parasites+in+vivo.">A flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo.</a> Malar J. 13 (1), 100 (2014).</li><li>Morohashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+abnormalities+in+the+spleen+of+an+mFtz-F1+gene-disrupted+mouse.">Structural and functional abnormalities in the spleen of an mFtz-F1 gene-disrupted mouse.</a> Blood. 93 (5), 1586-1594 (1999).</li><li>Shet, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+functional+platelet+abnormalities+in+Berkeley+sickle+cell+mice.">Morphological and functional platelet abnormalities in Berkeley sickle cell mice.</a> Blood Cells Mol Dis. 41 (1), 109-118 (2008).</li><li>Duraisingh, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+variation+of+Plasmodium+falciparum+merozoite+proteins+directs+receptor+targeting+for+invasion+of+human+erythrocytes.">Phenotypic variation of Plasmodium falciparum merozoite proteins directs receptor targeting for invasion of human erythrocytes.</a> EMBO J. 22 (5), 1047-1057 (2003).</li><li>Okoyeh, J. N., Pillai, C. R., Chitnis, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum+field+isolates+commonly+use+erythrocyte+invasion+pathways+that+are+independent+of+sialic+acid+residues+of+glycophorin+A.">Plasmodium falciparum field isolates commonly use erythrocyte invasion pathways that are independent of sialic acid residues of glycophorin A.</a> Infect Immun. 67 (11), 5784-5791 (1999).</li><li>Dolan, S. A., Miller, L. H., Wellems, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+switching+mechanism+in+the+invasion+of+erythrocytes+by+Plasmodium+falciparum.">Evidence for a switching mechanism in the invasion of erythrocytes by Plasmodium falciparum.</a> J Clin Invest. 86 (2), 618-624 (1990).</li></ol>

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>bisBenzimide H 33342 trihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>B2261</td>
			<td>Hoechst 33342. Store a 4mM stock solution at -20 &#176;C in distilled water</td>
		</tr>
		<tr>
			<td>Hoechst 34580</td>
			<td>Sigma-Aldrich</td>
			<td>63493</td>
			<td>Store a 2mM stock solution at -20 &#176;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 6mM stock solution at -20 &#176;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 2mg/ml stock solution at -20 &#176;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 25mg/ml stock solution at -20 &#176;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&#181;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

9.5K 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

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

フローサイトメトリーで固定し、生細胞におけるカルパイン活性の測定

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

免疫・感染学

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

フローサイトメトリーによって負傷した脊髄組織における免疫細胞の定量的評価：セルの精製方法のための新しい使用法

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

ヒトのインビトロ</em免疫バランスの初期状態の認識のためのスクリーニングツールとして>抑制

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

フローサイトメトリーによる胸腺正と負の選択の検討

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

フローサイトメトリーによるラットの腎臓からのマクロファージの表現型の特徴

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

網膜ミクログリアの貪食機能を評価します<em&gt;インビボ</em&gt;フローサイトメトリーベースのアッセイを使用しました

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

PM10における粒子結合のBet V 1アレルゲンのフローサイトメトリー分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

離とマウスとヒトの肺胞マクロファージの体外培養

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血によって人間の Monocyte のサブセットの解析フローのフローサイトメトリー解析

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

7 免疫細胞サブセット 2 螢光色素の差別フローサイトメトリー

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...