Vorremmo riconoscere George Cross per consigli generali sulla biologia trypanosome. Questo lavoro è stato supportato anche da un Bill e Melinda Gates Foundation GCE concessione per DS, un NSF Graduate Research Fellowship (DGE-1.325.261) per MRM e un NIH / NIAID (grant # AI085973) al FNP. Ringraziamo Galadriel Hovel-Miner per l&#39;uso del ceppo che contiene il sito del gene e il riconoscimento I-SCEI.

NOTA: Durante la procedura, è necessario mantenere le cellule in ghiaccio. supporti a freddo dovrebbero essere utilizzati in tutto. Raccolta 1. Esempio <ol><li> Grow Lister 427 tripanosomi ceppo sangue per una densità di 0,5 - 1 milione / ml. È meglio iniziare culture con un piccolo numero di parassiti. Spin down 50 x 10 6 cellule / campione per 10 min a 1500 x g. Assicurarsi di lasciare 1 x 10 6 cellule in coltura per poi utilizzare i controlli di anticorpi come positivi e negativi. NOTA: Questo protocollo può essere utilizzato anche su trypanosomes isolate dal sangue animale (vedi la discussione per i dettagli). </li><li> Pipetta o versare fuori la maggior parte del surnatante (lasciare circa 750 ml). Trasferire le cellule in una provetta da 1,5 ml microcentrifuga. </li><li> Spin cellule a 4 ° C per 4 min a 5200 xg in una microcentrifuga e rimuovere il surnatante. </li></ol> 2. L&#39;etichettatura magnetica <ol><li> Risospendere le cellule in 150 microlitri terreno di coltura (HMI-9 with siero per esempio) + anticorpo anti-VSG primaria alla diluizione appropriata. NOTA: L&#39;anticorpo anti-VSG è fatta in casa e utilizzato a una diluizione 1:50. È importante utilizzare un anticorpo primario anti-VSG che non è etichettato con un fluoroforo. </li><li> cellule Vortex utilizzando un vortice adattatore 60 a velocità 6 - 8 in una camera fredda a 4 ° C per 10 min. Lavare con 800 - 1.000 ml freddo HMI-9 con siero. </li><li> Spin a 4 ° C per 4 min a 5200 xg e aspirare il surnatante. Lavare con 1 ml di HMI-9 con il siero e spin come sopra. Aspirare il surnatante. Risospendere il pellet in 100 ml di HMI-9 con siero. </li><li> Aggiungere 110 ml di cellule magnetiche attivate ordinamento microsfere (MSC). Usa anti-topo, anti-coniglio, o perle anti-biotina come appropriato per l&#39;anticorpo anti-VSG primaria. Mescolare bene. cellule Vortex in una camera fredda a 4 ° C per 10 min. </li><li> Mentre le cellule sono vortex, impostare una separazione colonna / campione magnetico attivato su un magnete di separazione. Assicuratevi di avere un receptacle (usiamo una provetta da centrifuga da 15 ml) sotto la colonna per raccogliere il flusso continuo. Sistemare il dispositivo in una stanza fredda. </li><li> Aggiungere 2 ml di HMI-9 con il siero di colonna Prime. Dopo innesco posto un tubo da centrifuga da 15 ml sotto la colonna per raccogliere flusso continuo di ciascun campione. </li><li> Dopo il 10 min di incubazione nel passo 2.4, aggiungere 800-1000 ml freddo HMI-9 con siero. Spin a 4 ° C per 4 minuti a 5200g e rimuovere il surnatante per rimuovere l&#39;anticorpo non legato. Lavare con 1 ml di HMI-9 con siero e ripetere lasciando 100 ml di surnatante. </li><li> Flick la provetta vigorosamente ed ispezionare visivamente il tubo per assicurarsi che il pellet è completamente risospeso. Aggiungere 1 ml di HMI-9 con siero e pipetta su e giù per fare in modo che le cellule sono ben risospese. </li></ol> 3. La separazione magnetica <ol><li> Assicurarsi che le colonne sono preparati come descritto sopra. Applicare le cellule a colonna. Raccogliere flow-through con cellule esprimenti il ​​VS non dominanteG. 1 ml di terreno + trypanosomes prende tipicamente 6 - 7 min a fluire attraverso la colonna. </li><li> Lavare 2x con 1 ml di HMI-9 con siero raccogli flow-through nella stessa provetta come al punto 3.1. </li><li> Dividere flow-through (3 ml) in due provette da 1,5 ml microcentrifuga. NOTA: Questi possono essere combinati successivamente o mezzo possono essere utilizzati per isolare l&#39;RNA, etc. </li><li> Rimuovere colonna dal magnete. Aggiungere 3 ml di HMI-9 con il siero di colonna e immergersi colonna in un tubo da 15 ml centrifuga per ottenere cellule che esprimono la partenza, dominante VSG. Togliere 300 ml di materiale eluito per una successiva analisi. Conservare queste cellule in ghiaccio. </li><li> Per l&#39;uso come un segnale positivo e un controllo negativo, prendere circa 1 milione di cellule dalla cultura di partenza di cellule di controllo in crescita a 37 ° C (quelli che non prevede di cambiare frequentemente) e di pipetta in una provetta da 1,5 ml microcentrifuga. NOTA: cellule di controllo positive saranno colorate con l&#39;anticorpo fluoroforo marcato nel passaggio 4.1. Negativo cellule di controllo remanella macchia in tutto il resto del protocollo. </li><li> campioni Spin flow-through e campione di controllo degli anticorpi positivo a 4 ° C per 4 minuti a 5200g e rimuovere il surnatante. NOTA: Un&#39;altra 300 microlitri aliquota di cellule eluite può essere usato come controllo positivo per anti-VSG colorazione. </li></ol> 4. La colorazione per identificare contaminanti e commutatori <ol><li> campioni flow-through Risospendere e cellule di controllo positivo in 100 ml di HMI-9 con siero + primaria,, anti-VSG anticorpi fluoroforo marcato alla diluizione appropriata. NOTA: L&#39;anticorpo anti-VSG marcato è fatta in casa ed utilizzato a una diluizione di 1: 1000. Il fluoroforo marcato, anti-VSG può essere dalla stessa fonte, come l&#39;anticorpo non marcato nella separazione MACS step.There dovrebbe essere di 3 ml di campione a flusso continuo in due provette da microcentrifuga seguenti step 3.5. </li><li> Per analizzare tutte le flow-through mediante citometria di flusso, unire il pellet in due provette da microcentrifuga mentre re-susin attesa dei campioni in 100 ml di HMI-9 con siero di anticorpi anti-+ VSG. Mescolare bene. </li><li> cellule Vortex in una camera fredda a 4 ° C per 10 min. Aggiungere 800 - 1.000 ml freddo HMI-9 con siero. campioni Spin a 4 ° C per 4 minuti a 5200g e rimuovere il surnatante per rimuovere l&#39;anticorpo non legato. </li><li> Lavare le cellule con 1 ml di HMI-9 con siero. campioni Spin a 4 ° C per 4 minuti a 5200g e rimuovere il surnatante. Durante questo ultimo giro, spin down the-campioni eluiti dal punto 3.4 e il campione di controllo negativo. </li><li> Risospendere tutto il flusso-through e campioni eluiti in: 148,5 ml HMI-9 con siero, 25 microlitri conteggio sfere assoluto e 1,5 ml ioduro di propidio colorazione soluzione (175 ml in totale). Risospendere campioni di controllo degli anticorpi positivi e negativi in ​​175 ml HMI-9 con siero. Assicurarsi di registrare il numero di conteggio sfere assoluti / 25 ml per il lotto usati. </li><li> Eseguire tutti i campioni attraverso un citofluorimetro e raccogliere i dati. Le cellule morte macchiare come PI <sup&gt; +. Contaminanti macchia come PI - VSG +. Switcher macchia come PI - VSG - 8. </li><li> Calcola le frequenze con citometria a flusso di dati di commutazione. NOTA: Per le istruzioni su come calcolare frequenze di commutazione, si prega di fare riferimento al file supplementare <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54715/Flow_Cytometry_Calculations.docx" target="_blank">citometria a flusso Calculations.docx</a> e Tabella 1. </li></ol>

<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>

The authors have nothing to disclose.

Rispetto alla tecnica sperimentale, il componente più critico del protocollo è mantenere tutti i campioni fredda. Trypanosomes molto rapidamente internalizzare anticorpo legato alla loro superficie 9, ma questo processo è motilità dipendente, e non influenzano il dosaggio finché le cellule sono mantenuti a 4 ° C. Tutti i campioni devono essere sempre tenuti in ghiaccio, e pipettaggio dovrebbero essere fatte rapidamente per ridurre al minimo l&#39;esposizione all&#39;ambiente C di laboratorio di 25 °. F...

Il protozoo parassita, Trypanosoma brucei, è un agente eziologico di malattie che colpiscono gli esseri umani (via Trypanosoma brucei gambiense e Trypanosoma brucei rhodesiense) e animali (via Trypanosoma brucei brucei) in tutta l&#39;Africa sub-sahariana. Si trasmette all&#39;host mammiferi attraverso la saliva del vettore mosca tsetse. Sia umane che animali africani tripanosomiasi causano un grave onere economico in regioni endemiche, e pochi farmaci sono disponibili o in fase di sviluppo per il trattamento di entrambe le malattie. La comprensione dei meccanismi di evasione immunitaria è fondamentale per lo sviluppo di farmaci per la tripanosomiasi. Variazione antigenica del denso, Variante di superficie glicoproteina (VSG) mantello che copre il parassita è uno dei mezzi principali con cui T. brucei elude la risposta anticorpale dei mammiferi. Esistono circa 2.000 varianti del gene VSG nel genoma tripanosoma, ma solo uno è trascritto in un dato momento da una delle ~ 15 expressiti di Sion. &#39;Commutazione&#39; del VSG espresso può avvenire sia con la copia di un gene VSG nel sito espressione attiva, o attraverso l&#39;attivazione trascrizionale di un sito espressione precedentemente silenziosa (rivisto in 1). Anche se molto lavoro è stato dedicato alla comprensione variazione antigenica in T. brucei, i fattori che influenzano la frequenza di commutazione sono ancora poco conosciuti. Gli studi finora sono stati ostacolati dal fatto che, mentre la commutazione avviene stocasticamente in vitro, frequenze di commutazione sono molto bassi, dell&#39;ordine di 1 a circa 10 6 cellule 2. Questo rende difficile misurare se un fattore dato aumenta o diminuisce la frequenza di commutazione, poiché commutazione è difficile da rilevare in primo luogo. Prima del 2009, i metodi per isolare switchers in una data popolazione sono stati lungo e laborioso. Questi passaging trypanosomes attraverso topi immunizzati contro il VSG dominante e quindi che raccolgono le cellule inclusiil giorno dopo 3 o contando centinaia di cellule mediante immunofluorescenza 4,5. Un&#39;altra strategia si basa sulla selezione per la resistenza ai farmaci per isolare switcher 6. Perché trypanosomes africani coltivate in vitro sono in genere costituiti da una grande popolazione che esprime una variante importante, e una popolazione molto più piccola di switcher che esprimono varianti alternative, si fa riferimento alla grande variante in questo documento come dominante, VSG partenza. Così facendo, abbiamo in nessun modo vogliamo implicare che questo importante variante ha una maggiore fitness di altre varianti nella popolazione. Qui si descrive un metodo che può attendibilmente misurare il numero di trypanosomes esprimere un VSG non dominante in una data popolazione in 3 - 4 ore. Questo metodo è particolarmente utile quando si vuole verificare se un dato manipolazione genetica o trattamento farmacologico aumenta il numero di celle commutate in una popolazione. Invece di liberare la popolazione di cellule che esprimono il faiminant, a partire VSG attraverso le droghe o con mezzi immunologici, queste cellule vengono eliminati dal primo rivestimento con sfere magnetiche accoppiate ad anticorpi contro il VSG dominante e poi isolandoli su una colonna magnetica. La popolazione commutata viene poi raccolto in flusso continuo e trattata ancora con un fluoroforo anticorpo marcato anti-VSG per identificare contaminanti. La quantificazione è ottenuta aggiungendo un numero definito di conteggio sfere assoluti ad ogni campione in modo che il rapporto di perline di cellule può essere determinato e utilizzato per quantificare il numero di commutatori nella popolazione 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.

Il metodo descritto qui è stato utilizzato per dimostrare che le interruzioni doppio filamento all&#39;interno del sito espressione VSG aumentato il numero di switchers in una popolazione 8. Qui, vi mostriamo i risultati rappresentativi di una popolazione di tripanosomi che sono stati indotti in modo simile per generare un doppio filamento Break presso il sito di espressione. Confrontiamo questi trypanosomes a quelli che non sono stati indotti a generare una pausa doppio fila...

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.

rilevamento di<em&gt; Trypanosoma brucei</em&gt; Switching Variante superficie glicoproteina da magnetico cellulare Attivato Ordinamento e citometria a flusso

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

detection-trypanosoma-brucei-variant-surface-glycoprotein-switching

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

8.7K Views.  Rockefeller University. 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.

Video: 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)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Purificazione della popolazione di cellule specifiche per fluorescenza separazione delle cellule attivate (FACS)

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

Ricerca

Immunologia e infezioni

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 for telomerase for de novo telomere DNA synthesis and is therefore important for telomere maintenance. T. brucei is a protozoan parasite that causes sleeping sickness in humans and nagana in cattle. Once infected mammalian host, T. brucei cell regularly switches its surface antigen to evade the host's immune attack. We have recently demonstrated that the T. brucei telomere structure plays an essential role in regulation of surface antigen gene expression, which is critical for T. brucei pathogenesis. However, T. brucei telomere structure has not been extensively studied due to the limitation of methods for analysis of this specialized structure. We have now successfully adopted the native in-gel hybridization and ligation-mediated primer extension methods for examination of the telomere G-overhang structure and an adaptor ligation method for determination of the telomere terminal nucleotide in T. brucei cells. Here, we will describe the protocols in detail and compare their different advantages and limitations.

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

Telomeres are essential for chromosome stability and the telomere G-overhang structure is essential for telomerase-mediated telomere maintenance. We have recently adopted two methods for detecting the telomere G-overhang structure in Trypanosoma brucei, which are native in-gel hybridization and ligation-mediated primer extension, which will be described.

L&#39;esame dei telomeri G-Struttura in aggetto Trypanosoma brucei</em

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 cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Analisi Citometria a flusso di cellule immunitarie all&#39;interno aorte Murine

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

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.

Esame di Selezione timica positivi e negativi per Citometria a Flusso

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

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Rilevamento di nanoparticelle fluorescenti Interazioni con Primaria immunitario sottopopolazioni cellulari mediante citometria di flusso

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

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

High-throughput di Gene Tagging in<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

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.

Caratterizzazione fenotipica dei macrofagi dal rene di ratto mediante citometria di flusso

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

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Separazione delle cellule fluorescenza-attivato per la purificazione delle cellule dendritiche plasmacitoidi dal midollo osseo di topo

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

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

Misura della T cellulare alloreattività Utilizzando Imaging Citometria a Flusso

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

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.

Isolamento e coltura In Vitro dei macrofagi alveolari murini ed umani

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

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