Name: detection of trypanosoma brucei variant surface glycoprotein switching by magnetic activated cell sorting and flow cytometry
Uploaded: 2016-10-19T15:00:00
Description: Watch this Scientific Journal Video about Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry at JoVE.com

Vi vil gerne anerkende George Cross til generel rådgivning om trypanosominfektion biologi. Dette arbejde blev også støttet af en Bill og Melinda Gates Foundation GCE tilskud til DS, en NSF Graduate Research Fellowship (DGE-1.325.261) til MRM og en NIH / NIAID (tilskud # AI085973) til FNP. Vi takker Galadriel Hovel-Miner til anvendelse af stamme indeholdende I-SCEI genet og genkendelsessted.

BEMÆRK: I hele den procedure, er det nødvendigt at holde celler på is. Kolde medier bør også anvendes overalt. 1. Prøve Høst <ol><li> Grow Lister 427 stamme blodbanen trypanosomer til en densitet på 0,5 - 1 million / ml. Det er bedst at starte kulturerne med et lille antal parasitter. Spin down 50 x 10 6 celler / prøve i 10 minutter ved 1.500 x g. Vær sikker på at efterlade 1 x 10 6 celler i kultur til senere brug som positive og negative antistof kontrolle...

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

Med hensyn til eksperimentel teknik, er den mest kritiske komponent af protokollen holde alle prøverne kolde. Trypanosomer meget hurtigt internalisere antistof bundet til deres overflade 9, men denne proces er motilitet afhængig, og påvirker ikke assayet, så længe cellerne holdt ved 4 ° C. Alle prøver skal altid holdes på is, og pipettering bør ske hurtigt for at minimere eksponering for 25 ° C lab miljø. Kolde HMI-9 med serum bør være tilgængelige i starten af ​​forsøget, og bør holdes p?...

Den protozoparasit, Trypanosoma brucei, er en agens af sygdomme, der påvirker mennesker (via Trypanosoma brucei gambiense og Trypanosoma brucei rhodesiense) og dyr (via Trypanosoma brucei brucei) i hele Afrika syd for Sahara. Den overføres til pattedyrværten gennem spyt af tsetsefluen vektor. Både menneskers og dyrs afrikanske trypanosomiasis forårsage en alvorlig økonomisk byrde i endemiske områder, og få lægemidler er tilgængelige eller under udvikling til behandling enten sygdom. Forståelse mekanismer immun unddragelse er afgørende for udviklingen af ​​lægemidler til trypanosomiasis. Antigen variation af den tætte, Variant Surface (VSG) pels, der dækker parasitten er en af de primære måder, hvorpå T. brucei undviger den mammale antistofrespons. Ca. 2.000 varianter af VSG-genet findes i trypanosominfektion genomet, men kun én transkriberes til enhver tid fra en af ~ 15 expressionens sites. &quot;Switching&quot; af udtrykte VSG kan ske enten ved at kopiere en VSG gen i det aktive udtryk site, eller ved transkriptionel aktivering af en tidligere tavs udtryk websted (revideret i 1). Selvom meget arbejde har været afsat til at forstå antigen variation i T. brucei, de faktorer, der påvirker skifte frekvens er stadig dårligt forstået. Undersøgelser til dato er blevet vanskeliggjort af, at mens skift sker stokastisk in vitro, skifte frekvenser er meget lave, i størrelsesordenen 1 i omkring 10 6 celler 2. Dette gør det vanskeligt at måle, om en given faktor stiger eller falder skifte frekvens, da skift er svært at opdage i første omgang. Før 2009, metoder til isolering omskiftere i en given population var langvarig og arbejdskrævende. Disse omfattede passage trypanosomer gennem mus immuniseret mod den dominerende VSG og derefter høste celleren dag senere 3, eller tælle hundredvis af celler ved immunofluorescens 4,5. En anden strategi er afhængig af selektion for resistens at isolere switchers 6. Fordi afrikanske trypanosomer dyrket in vitro er typisk består af en stor befolkning udtrykker en større variant, og en meget mindre population af switchers udtrykker alternative varianter, henviser vi til den store variant hele dette papir som den dominerende, startende VSG. Dermed vi ikke på nogen måde ønsker at antyde, at denne store variant har større kondition end andre varianter i populationen. Her beskriver vi en metode, som pålideligt kan måle antallet af trypanosomer udtrykker et ikke-dominerende VSG i en given population i 3 - 4 timer. Denne fremgangsmåde er særlig nyttig til, når man ønsker at konstatere, om en given genetisk manipulation eller lægemiddelbehandling øger antallet af koblede celler i en population. I stedet for at befri populationen af ​​celler, der udtrykker dominant startede VSG gennem narkotika eller ved immunologiske midler, er disse celler elimineres ved først at overtrække dem med magnetiske perler koblet til antistof mod den dominerende VSG og derefter isolere dem på en magnetisk søjle. Det koblede population opsamles derefter i gennemløbet og farvet igen med en fluorofor mærket anti-VSG-antistof til at identificere forureninger. Kvantificering opnås ved at tilsætte et defineret antal absolutte optælling perler til hver prøve, således at kan bestemmes forholdet mellem perler og celler og anvendes til at kvantificere antallet af omskiftere i populationen 7.

<table border="0" cellpadding="0" cellspacing="0" width="644">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">unlabeled anti-VSG antibody</td>
			<td style="width:143px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:167px;">VSG antibody is made in house</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">fluorophore labeled anti-VSG antibody</td>
			<td style="width:143px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:167px;">VSG antibody is made in house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HMI-9 media</td>
			<td style="width:143px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:167px;">HMI-9 is made in house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Propidium Iodide</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td>556463</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CountBright absolute counting beads</td>
			<td style="width:143px;">Thermofisher Scientific</td>
			<td>C36950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LD columns</td>
			<td style="width:143px;">Miltenyi Biotech</td>
			<td>130-042-901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACS magnet</td>
			<td style="width:143px;">Miltenyi Biotech</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACS magnetic separator</td>
			<td style="width:143px;">Miltenyi Biotech</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">vortex adapter-60</td>
			<td style="width:143px;">ThermoFisher scientific</td>
			<td>AM10014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">flow cytometer</td>
			<td style="width:143px;">Coulter</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">flow cytometer analysis software</td>
			<td style="width:143px;">FloJo</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

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

Den her beskrevne metode blev anvendt til at påvise, at dobbelt-strenget pauser i VSG udtryk stedet øget antallet af omskiftere i en population 8. Her viser vi repræsentative resultater fra en population af trypanosomer, som er blevet tilsvarende induceres til at frembringe en dobbelt-strenget pause på ekspressionsstedet. Vi sammenligner disse trypanosomer til dem der ikke er blevet induceret til at generere en dobbeltstrenget brud. Figur 1 viser repræsen...

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

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

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

påvisning af<em&gt; Trypanosoma brucei</em&gt; Variant overfladeglycoprotein Skift af magnetisk cellesortering og flowcytometri

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

Research

JoVE Journal

Immunology and Infection

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

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

Undersøgelse af tymisk positiv og negativ selektion ved flowcytometri

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

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Påvisning af fluorescerende Nanopartikel Interaktioner med Primary Immune cellesubpopulationer ved flowcytometri

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

High-throughput Gene Tagging in Trypanosoma brucei

High-throughput Gene Tagging in Trypanosoma brucei

High-throughput Gene Tagging i<em&gt; Trypanosoma brucei</em

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

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

Fænotypisk karakterisering af makrofager fra rottenyre ved flowcytometri

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

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescens-aktiveret cellesortering til oprensning af Plasmacytoide dendritiske celler fra knoglemarven i mus

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

Måling af T-celle alloreaktivitet Brug Imaging flowcytometri

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation og In Vitro kultur af Murine og menneskelige alveolær makrofager

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