Name: in vivo assessment of rodent plasmodium parasitemia and merozoite invasion by flow cytometry
Uploaded: 2015-04-05T14:00:00
Description: Watch this Scientific Journal Video about In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry at JoVE.com

ونحن نعترف تمويل الدعم من مجلس البحوث الصحية والطبية الوطنية (منح APP605524، 490037 1047082 و)، ومجلس الأبحاث الأسترالي (منح DP12010061)، والاستراتيجية البنية التحتية البحوث التعاونية الوطنية في أستراليا وصندوق الاستثمار التعليم من قسم الابتكار والصناعة والعلوم والبحوث. حزب الرابطة الاسلامية هي المستفيدة من جائزة الدراسات العليا الاسترالية.

وأجريت جميع الإجراءات وفقا لسياسات جامعة ماكواري ويتفق مع المجلس الوطني للصحة والبحوث الطبية (NHMRC) رمز الاسترالي من الممارسة. تم إجراء العمل في إطار أخلاقيات اتفاق لا ARA 2012/017 الموافقة والحصول عليها من لجنة الأخلاقيات الحيوانية في جامعة ماكواري. أجريت جميع التجارب على الفئران SJL ...

<ol>
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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. 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وصفناها طريقة لقياس كل من الغزو الطفيل وأقسومة العينات في الجسم الحي. من حيث قياس الطفيل، وهذه الطريقة يوفر ميزة على الطرق السابقة 10-13 في ذلك HJ-كرات الدم الحمراء يمكن تمييزها عن pRBCs، مما يقلل من عدد من الأحداث الإيجابية الكاذبة. بينما HJ-كرات الدم الحمراء وعا...

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.

قياس الطفيل. لقياس الطفيل، ينبغي أولا أن يتم اختيار خلايا الدم، والضوضاء، والحطام والصفائح الدموية استبعاد، على أساس FSC / خصائص التعاون بين بلدان الجنوب (الشكل 2A). اعتمادا على عداد الكريات المستخدمة، وينبغي بعد ذلك يتم اخت...

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

ويغزو الطفيلي الملاريا ومكررات داخل خلايا الدم الحمراء. تقييم دقيق للغزو أقسومة والطفيل هو بالتالي حاسما في تقييم مسار عدوى الملاريا. نحن هنا وصف التدفق الخلوي بروتوكول أساس لقياس هذه المعايير في نموذج الفأر من الملاريا.

في تقييم فيفو من القوارض المتصورة الطفيل وأقسومة الغزو من قبل التدفق الخلوي

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

Research

JoVE Journal

Immunology and Infection

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

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

قياس نشاط خلايا في Calpain الثابتة والعيش من خلال التدفق الخلوي

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

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

التقييم الكمي لخلايا المناعة في الأنسجة النخاع الشوكي المصاب بواسطة التدفق الخلوي : رواية عن استخدام أسلوب خلية تنقية

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

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

الإنسان في المختبر قمع وأداة الكشف عن الاعتراف للدولة في وقت مبكر من الخلل المناعي

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

دراسة التوتة اختيار الإيجابية والسلبية من قياس التدفق الخلوي

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

التوصيف المظهري لالضامة من الكلى الجرذ بواسطة التدفق الخلوي

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

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

تقييم الشبكية دبقية أكلة وظيفة<em&gt; في فيفو</em&gt; باستخدام الفحص القائمة على الخلوي التدفق

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

تدفق تحليل Cytometric من متجهة الجسيمات بيت ت 1 مسببات الحساسية في 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

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

العزلة والثقافة في المختبر من البلاعم السنخية مورين والبشرية

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

وصف مجموعات فرعية الوحيدات البشرية بالدم كله يتدفق التحليل الخلوي

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

التمييز من سبع مجموعات فرعية الخلايا المناعية قبل يومين-fluorochrome التدفق الخلوي

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