Wir erkennen die Finanzierung der Unterstützung des National Health und Medical Research Council (gewähren APP605524, 490.037 und 1.047.082), der Australian Research Council (gewähren DP12010061), der National Collaborative Research Infrastructure Strategy of Australia und der Bildung Investmentfonds von der Abteilung Innovation, Industrie , Wissenschaft und Forschung. PML ist ein Empfänger einer australischen Postgraduate Award.

Alle Verfahren wurden in Übereinstimmung mit den Richtlinien der Macquarie University durchgeführt und entsprach der nationalen Gesundheits-und Medical Research Council (NHMRC) Australian Verhaltenskodex. Die Arbeit wurde im Rahmen des Abkommens Ethik Kein ARA 2012/017 genehmigt und von der Tierethikkommission an der Macquarie Universität erhalten durchgeführt. Alle Experimente wurden an SJL / J-Mäusen durchgeführt, sofern nicht anders angegeben. 1. Mäuse und experimentelle Malariainfektion <ol><li> Hausmäuse bei kontrollierter Temperatur (21 ° C) mit einem 12:12 Stunden Licht-Dunkel-Zyklus. </li><li> Tauwetter ein Aliquot von kryokonservierten P. chabaudi adami DS mit 5% parasitierten Erythrozyten (PRBCs) und injizieren 200 ul in die Bauchhöhle von C57BL / 6 Spendermaus. </li><li> Überwachen Spendermaus Parasitämie alle 1 - 2 Tage mittels Durchflusszytometrie, wie in Abschnitt 3 beschrieben - 4 dieses Protokolls. Sobald Spendern erreicht 5 - 15% Parasitämie, sammeln Blut durch Herzpunktion wie folgt: <ol><li>Aspirat 100 ul Heparin (300 U / ml Heparin in der Maus Ringer-Lösung (MTR) (154 mM NaCl, 5,6 mM KCl, 1 mM MgCl 2, 2,2 mM CaCl 2, 20 mM HEPES, 10 mM Glucose, 30 U / ml Heparin, pH 7,4, 0,22 um Filter sterilisiert)) in eine 1 ml Spritze mit einer 26 G-Nadel. </li><li> Anesthetize die Maus durch Inhalation mit 5% Isofluoran bestätigte die gewünschte Tiefe der Anästhesie durch Fehlen Pedalrückzugsreaktion und Hornhautreflexe. </li><li> Platzieren der Maus auf die Seite und senkrecht Einstichkanüle gerade unterhalb des Ellenbogens, durch die Rippen und in das Herz. Langsam herausziehen Spritzenkolben und drehen Nadel bis 0,5-1 ml Blut erhalten wird. </li><li> Führen humane Sterbehilfe durch Genickbruch unter Narkose. </li></ol></li><li> Berechne die prbcS pro Volumen von Spenderblut Annahme einer Blutbild von 9 x 10 6 RBC / ul (dh, wenn der Spender mit 10% Parasitämie bei geopfert Blut 9 x 10 5 enthältpRBC / ul). Verdünne parasitierten Blut in Krebs-gepufferter Salzlösung (126 mM NaCl, 2,5 mM KCl, 25 mM NaHCO 3, 1,2 mM NaH 2 PO 4, 1,2 mM MgCl 2, 2,5 mM CaCl 2, 0,2% Glucose, pH 7,2, 0,22 um Filter sterilisiert) so dass die Konzentration 1 x 10 4 prbcS pro 200 &amp; mgr; l und 200 &amp; mgr; l Injektion in die Bauchhöhle von Mäusen für die RBC Kennzeichnung und Transfusionsexperiment erforderlich. </li><li> Überwachen Parasitämie alle 1 - 2 Tage mittels Durchflusszytometrie als in den Abschnitten 3 beschrieben - 4 dieses Protokolls. </li></ol> 2. Markierung der Erythrozyten und Transfusions <ol><li> Sammeln heparinisiertem Blut von Mäusen durch Herzpunktion, wie in Schritt 1.3 beschrieben, und legen Sie auf dem Eis. Halten Blut bei 4 ° C zu allen Zeiten. Sammeln etwa 200 ul Blut pro Maus injiziert werden. Bei Bedarf aufgeteilt Blut in zwei Proben und Behandlung einer Probe, wie gewünscht. HINWEIS: Auf diese Weise Invasion der behandelten sample kann zu einer Kontrolle verglichen werden, unbehandelten Probe (siehe Abbildung 1 schematisch). </li><li> Bereiten Sie eine 2x Lösung von jeder Leuchtstoff RBC-Label. <ol><li> Verdünne eine 2 mg / ml Stammlösung von Atto 633-N-Hydroxysuccinimid (Atto 633-NHS) in MTR so daß die Konzentration 20 ug / ml. </li><li> Verdünne mit 25 mg / ml Vorratslösung von Biotin-N-hydroxysuccinimidester (Biotin-NHS) in MTR so daß die Konzentration 250 ug / ml. </li></ol></li><li> Aufgeteilt Blutproben in zwei Röhrchen entnommen und das gleiche Volumen von 2x Atto 633-NHS-Lösung auf ein Rohr, und ein gleiches Volumen von 2x-Biotin-NHS zur anderen Röhre. Mischen Sie sofort. Inkubieren Blut bei 4 ° C für 1 h mit konstantem langsamem Mischen oder alternativ mischen Probe jedes 10-15 min. </li><li> Blut beschriftet 3 Mal Waschen mit MTRC (MTR + 0,5% Rinderserumalbumin (BSA), 0,22 um Filter sterilisiert) wie folgt: <ol><li> Fügen Sie mindestens 2 Volumina MTRC, Zentrifuge bei 750 xg für 5 min, entfernen Sie den Überstand und resuspend die Pellets in mindestens 2 Volumina MTRC. Wiederholen Sie 2 weitere Male. </li><li> Nach dem letzten Waschen, Zentrifugieren bei 750 × g für 5 min pelletiert und verbinden das Blut in verschiedenen Kombinationen (dh unbehandelten Atto 633 / Biotin behandelten, unbehandelten Biotin / behandelt Atto 633). Hinzufügen MTR bis zu einem Volumen von 200 ul pro Maus zu machen, eingespritzt werden. </li></ol></li><li> Injizieren beschriftet Blut intravaskulär in Nagetier Malaria infizierten Mäusen oder infizierten Kontrollen wie folgt: <ol><li> Führen Trans bei 2 - 15% Parasitämie am Gipfel des Parasiten Schizonten. HINWEIS: In diesem Fall etwa in der Mitte durch die Dunkel-Zyklus (dh zwischen 12 bis 2 Uhr morgens an einem normalen Lichtzyklus oder 12-2 Uhr an einem umgekehrten Lichtzyklus). </li><li> Warm-Mäuse mit einer Wärmelampe für 5-10 Minuten, um den Blutfluss zu erhöhen, aber keine Vorkehrungen, um die Mäuse überhitzen. Zeigen Mäuse in einem Rückhalteeinrichtung und intravaskulär injizieren 200 ul markierte Blut (ca. 1 - 2 x 10 9 RBCs) über die Schwanzvene. </li></ol></li><li> Sammeln von Blutproben zu verschiedenen Zeitpunkten nach der Injektion, wie in Abschnitt 3 beschrieben. HINWEIS: Invasion Raten von weniger als 10 min nach der Injektion quantitativ bestimmt werden, abhängig von Parasitämie der Empfängermaus. </li></ol> 3. Entnahme von Blutproben und Vorbereitung für die Durchflusszytometrie <ol><li> Vorbereitung 50 ul Farblösung pro Probe plus zusätzliche, am Tag des Experiments wie folgt: <ol><li> Abtauen eines Aliquots von 6 mM JC-1 bei -20 ° C in Dimethylsulfoxid (DMSO) gelagert. </li><li> Warm 50 ul MTRC pro Probe, plus extra, auf 37 ° C. </li><li> Während kontinuierlich Vortexen des vorgewärmten MTRC Fügen JC-1, so dass die Endkonzentration 12 &amp; mgr; M. Dies wird gemacht, um die Bildung von JC-1-Aggregate zu reduzieren; wenn Sie bilden Aggregate, nicht Zentrifugenröhrchen, da dies Färbung zu verhindern. ANMERKUNG: Eine kleine Menge von Aggregation hat keinen Einfluss auf Ergebnisse. </li><li> Anti hinzufügenCD45 APC eFluor 780 und Anti-CD71 PerCP eFluor 710, so dass die Endkonzentration 1 ug / ml. Bei Durchführung des markierten RBC Experiment hinzuzufügen Streptavidin PE-Cy7, so daß die Endkonzentration 1 ug / ml. </li></ol></li><li> Führen Schwanz Blutungen durch Amputation der Schwanzspitze oder Platzwunde des Blutgefäßes in den Schwanz. Geben Sie einen Tropfen Blut Schwanz auf eine kleine wiegen Boot oder Glasträger. Nach der Blutentnahme, sicherzustellen, dass die Blutung aufgehört hat. Pipette 3 ul Schwanzblut in 50 &amp; mgr; l Färbelösung auf 37 ° C vorgewärmt und Heizen von Proben bei 37 ° C für 20 min. </li><li> Abtauen eines Aliquots 4 mM Hoechst 33342 bei -20 ° C in destilliertem Wasser (bei Verwendung eines 355 nm Laser) gespeichert oder 2 mM Hoechst 34580 bei -20 ° C in destilliertem Wasser (bei Verwendung eines 405 nm Laser) gespeichert. Bereiten Sie 500 ul pro Probe, plus extra, einer 4 &amp; mgr; M oder 2 uM Lösung von Hoechst in MTRC auf. </li><li> Fügen Sie 500 ul von 4 uM Hoechst33.342 oder 2 uM Hoechst 34.580 Proben und Inkubation bei Raumtemperatur für 20 min. </li><li> Zentrifuge Zellen bei 750 g für 3 min bei 4 ° C, Überstand verwerfen, fügen 700 ul MTRC und durch Strömungs analysieren Zytometrie. </li></ol> 4. Durchflusszytometrie <ol><li> Analysieren Sie die Proben mit einem 355/488/633 nm Laser oder 405/488/633 nm Lasergerät. </li><li> Filter Proben durch einen 35 um Zellsieb unmittelbar vor der Analyse. Spülen Zellsieb mit destilliertem Wasser zwischen den Proben und Wiederverwendung. </li><li> Nehmen Sie genug Ereignisse, so dass die kleinste Bevölkerung mindestens 500 Ereignisse (in der Regel eine Million - 10 Millionen Gesamt Ereignisse pro Probe). </li><li> Excite Hoechst 33342 mit einem 355-nm-Laser und erkennt Ereignisse durch eine 460/50-Filter. Excite Hoechst 34580 mit einem 405-nm-Laser und erkennt Ereignisse durch eine 460/50-Filter. Excite JC-1, anti-CD71 PerCP eFluor 710 und Streptavidin PE-Cy7 mit einem 488-nm-Laser und erkennt Ereignisse durch ein 530/40, 692/40 und 750LP filtern jeweiligenly. Excite Atto 633 und Anti-CD45 APC eFluor 780 mit einem 633-nm-Laser und erkennt Ereignisse durch ein 670/30 oder 750LP-Filter auf. </li><li> Führen Sie die Analyse und Kompensation mit geeigneten Durchflusszytometrie-Analyse-Software wie folgt: <ol><li> Wählen ganze Zellen und verstehen sich inklusive Lärm, Schmutz und Blutplättchen basierend auf FSC / SSC Eigenschaften (Abbildung 2A). Auswahl einzelner Zellen, die entweder auf Triggerimpulsbreite (2B) oder mit dem FSC-Peakfläche zu Höhe Verhältnis (2C). </li><li> Wählen reifen Erythrozyten und schließen RBC Vorläuferzellen und Leukozyten, basierend auf negativen Fluoreszenz in den PerCP eFluor 710 und APC eFluor 780 Kanäle (2D). </li><li> Beim Arbeiten mit der Bezeichnung RBC Experiment, wählen Sie die einzelnen Population markierter Erythrozyten auf Basis von PE-Cy7 und Atto 633 Fluoreszenz und analysieren diese separat (3A). </li><li> Wählen Sie basierend auf positive Fluoreszenz sowohl für JC-1 und Hoechst PRBCs ( <strong&gt; 2E-H). </li></ol></li></ol> 5. Berechnungen und Statistik <ol><li> Berechnen Parasitämie durch Dividieren der Anzahl von prbcS durch die Gesamtzahl von Erythrozyten (dh die Anzahl von Ereignissen in Gate Q2 in Gatters G4 geteilt durch die Anzahl von Ereignissen). Bei der Durchführung des Invasionsassay Parasitämie der beiden markierten Populationen, indem die Anzahl der markierten prbcS durch die Anzahl der markierten RBCs in der Population (berechnet, dh, die Anzahl der Ereignisse, die in beide Tore L1 und Q2 sind, dividiert durch die Anzahl der Ereignisse, im Tor L1). HINWEIS: Labeled Parasitämie variiert beträchtlich in Abhängigkeit von Faktoren wie beispielsweise die endogene Parasitämie und Anzahl zirkulierender Merozoiten. Aus diesem Grund ist es hilfreich, die Parasitämie Verhältnis, das als das Parasitämie des behandelten markierten Population in jeder einzelnen Maus, geteilt durch die Parasitämie der Steuer markierten Population berechnete melden. Um mögliche Farbeffekte zu korrigieren, führt zu einem Berichts eine durchschnittliche Parasitämie Verhältnis zwischen den beiden Farbstoffkombinationen. </li><li> Bestimmen Sie eine statistische Signifikanz von Verhältnissen mit dem eine Probe t-Test mit 1 als hypothetische Mittelwert. </li></ol>

<ol>
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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>

Die Autoren haben nichts zu offenbaren.

Wir haben ein Verfahren für die gleichzeitige Messung von Parasitämie und Merozoiten Invasion in vivo Proben beschrieben. In Bezug auf die Parasitämie Messung bietet dieses Verfahren einen Vorteil gegenüber bisherigen Methoden 10-13 in diesem HJ-Erythrozyten aus PRBCs unterschieden werden, wodurch die Anzahl der falsch-positive Ereignisse zu reduzieren. Während HJ-RBCs sind gewöhnlich beim Menschen selten einige Studien berichten hohen Niveaus in Mäusen 15,16 die Unterscheidung zwis...

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.

Die Messung der Parasitämie. Für die Messung der Parasitämie sollte Blutkörperchen zuerst ausgewählt werden, und Lärm, Schmutz und Plättchen ausgeschlossen, bezogen auf FSC / SSC-Eigenschaften (2A). Abhängig von der Zytometer verwendet wird, sollten einzelne Zellen dann basierend auf jeder Triggerimpulsbreite (2B) oder FSC Peakhöhe auf Flächenverhältnis (2C) ausgewählt werden. Verbleibende Ereignisse sollten von Leukozyten bestehen,...

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

Die Malaria-Parasiten dringt und Wiederholungen in den roten Blutkörperchen. Die genaue Beurteilung der Merozoiten Invasion und Parasitämie ist daher entscheidend für die Beurteilung der Verlauf der Malaria-Infektion. Hier beschreiben wir eine Durchflusszytometrie basiertes Protokoll für die Messung dieser Parameter in einem Maus-Modell der Malaria.

In-vivo-Bewertung der Nager Plasmodium Parasitämie und Merozoite Invasion durch Durchflusszytometrie

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 Aufrufe.  Macquarie University.  Die Malaria-Parasiten dringt und Wiederholungen in den roten Blutkörperchen. Die genaue Beurteilung der Merozoiten Invasion und Parasitämie ist daher entscheidend für die Beurteilung der Verlauf der Malaria-Infektion. Hier beschreiben wir eine Durchflusszytometrie basiertes Protokoll für die Messung dieser Parameter in einem Maus-Modell der Malaria. 

Serie: 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.

Mess-Calpain Aktivität in fixierten und lebenden Zellen mittels Durchflusszytometrie

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

Forschung

Immunologie und Infektion

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.

Quantitative Beurteilung der Immunzellen in das verletzte Rückenmark Tissue mittels Durchflusszytometrie: eine neue Verwendung für eine Zelle Reinigungsverfahren

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.

Mensch In Vitro Suppression als Screening-Tool für die Anerkennung von 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 ...

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.

Prüfung Thymic positive und negative Selektion mittels Durchflusszytometrie

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.

Phänotypische Charakterisierung von Makrophagen aus Rattenniere mittels Durchflusszytometrie

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.

Die Beurteilung Retinal Microglial Phagozytische Funktion<em&gt; In Vivo</em&gt; Mit einem Durchfluss-Zytometrie basierten Assay

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.

Durchflusszytometrische Analyse von partikelgebundenen Bet v 1 Allergens 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 ...

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.

Isolation und In-vitro- Kultur von murinen und menschlichen alveoläre Makrophagen

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).

Charakterisierung der menschliche Monocyte Teilmengen von Vollblut Flow Cytometry Analysis

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

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

Vorbereitung des ganzen Knochenmarks für die Mass Cytometry Analyse von Neutrophilen-Linienzellen

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...