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://www.jove.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/pubmed?term=((Antigenic+variation+in+African+trypanosomes%5BTitle%5D)+AND+%22Molecular+%26+Biochemical+Parasitology%22%5BJournal%5D)">Antigenic variation in African trypanosomes.</a> Molecular & 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/pubmed?term=((Analysis+of+antigen+switching+rates+in+Trypanosoma+brucei%5BTitle%5D)+AND+%22Parasitology%22%5BJournal%5D)">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/pubmed?term=((Telomere+exchange+can+be+an+important+mechanism+of+variant+surface+glycoprotein+gene+switching+in+Trypanosoma+brucei%5BTitle%5D)+AND+%22Mol+Biochem+Parasitol%22%5BJournal%5D)">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/pubmed?term=((High+frequency+of+antigenic+variation+in+Trypanosoma+brucei+rhodesiense+infections%5BTitle%5D)+AND+%22Parasitology%22%5BJournal%5D)">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/pubmed?term=((Analysis+of+antigenic+types+appearing+in+first+relapse+populations+of+clones+of+Trypanosoma+brucei%5BTitle%5D)+AND+%22Parasitology%22%5BJournal%5D)">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/pubmed?term=((Analysis+of+Trypanosoma+brucei+vsg+expression+site+switching+in+vitro%5BTitle%5D)+AND+%22Mol+Biochem+Parasitol%22%5BJournal%5D)">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/pubmed?term=((A+histone+methyltransferase+modulates+antigenic+variation+in+African+trypanosomes%5BTitle%5D)+AND+%22PLoS+Biol%22%5BJournal%5D)">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/pubmed?term=((A+yeast-endonuclease-generated+DNA+break+induces+antigenic+switching+in+Trypanosoma+brucei%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Hydrodynamic+Flow-Mediated+Protein+Sorting+on+the+Cell+Surface+of+Trypanosomes%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((The+in+vivo+dynamics+of+antigenic+variation+in+Trypanosoma+brucei%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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><tbody><tr><td>unlabeled anti-VSG antibody</td><td>n/a</td><td>n/a</td><td>VSG antibody is made in house</td></tr><tr><td>fluorophore labeled anti-VSG antibody</td><td>n/a</td><td>n/a</td><td>VSG antibody is made in house</td></tr><tr><td>HMI-9 media</td><td>n/a</td><td>n/a</td><td>HMI-9 is made in house</td></tr><tr><td>Propidium Iodide</td><td>BD Pharmingen</td><td>556463</td><td></td></tr><tr><td>CountBright absolute counting beads</td><td>Thermofisher Scientific</td><td>C36950</td><td></td></tr><tr><td>LD columns</td><td>Miltenyi Biotech</td><td>130-042-901</td><td></td></tr><tr><td>MACS magnet</td><td>Miltenyi Biotech</td><td>130-042-303</td><td></td></tr><tr><td>MACS magnetic separator</td><td>Miltenyi Biotech</td><td>130-042-302</td><td></td></tr><tr><td>vortex adapter-60</td><td>ThermoFisher scientific</td><td>AM10014</td><td></td></tr><tr><td>flow cytometer</td><td>Coulter</td><td>n/a</td><td></td></tr><tr><td>flow cytometer analysis software</td><td>FloJo</td><td>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

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

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer1-7. In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens7. It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to na&iuml;ve counterparts7-10. However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression11,12. Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo.
Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to na&iuml;ve MDSC 13. MDSC percentages are significantly less in lymphoid compartments of na&iuml;ve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1+ leukocytes from na&iuml;ve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1+ leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1+ leukocytes from spleens of na&iuml;ve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas na&iuml;ve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from na&iuml;ve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1+ leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1+ enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (na&iuml;ve vs. tumor- bearing) in in vivo and in vitro assays.

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

This is a rapid and comprehensive method of immunophenotyping Myeloid Derived Suppressor Cells (MDSC) and enriching Gr-1+ leukocytes from mouse spleens. This method uses flow cytometry and AutoMACS Cell Sorting to enrich for viable Gr-1+ leukocytes prior to FACS sorting of MDSC for use in vivo and in vitro assays.

Preparazione di cellule derivate mieloidi soppressore (MDSC) da Naive e Pancreatiche topi affetti da tumore in citometria a flusso e di smistamento automatico magnetico cellulare Attivato (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

Ricerca

Medicina

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Utilizzo di proteine ​​fluorescenti per monitorare Glycosome Dynamics in Tripanosoma africano

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Immunologia e infezioni

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.

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

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of malaria.

In Vivo Valutazione del roditore Plasmodium parassitemia e merozoite Invasion di Citometria a flusso

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

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

Rapida rilevazione omogenea dei saggi biologici Utilizzando Magnetic biopercezione sistema di modulazione

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Biologia

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 ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

Flusso Virometry per analizzare antigenica spettri di virioni e extracellulare vescicole

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

Rilevamento e l&#39;arricchimento di Rare antigene-specifiche cellule B per l&#39;analisi del fenotipo e funzione

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns)

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is the best method for diagnosis of first stage HAT and furthermore this parasite purification method permits serological and research investigations.
HAT is caused by Tsetse fly transmitted Trypanosoma brucei gambiense and T. b. rhodesiense. Related trypanosomes are the causative agents of animal trypanosomiasis. Trypanosome detection is essential for HAT diagnosis, treatment and follow-up. The technique described here is the most sensitive parasite detection technique, adapted to field conditions for the diagnosis of T. b. gambiense HAT and can be completed within one hour. Blood is layered onto an anion-exchanger column (DEAE cellulose) previously adjusted to pH 8, and elution buffer is added. Highly negatively charged blood cells are adsorbed onto the column whereas the less negatively charged trypanosomes pass through. Collected trypanosomes are pelleted by centrifugation and observed by microscopy. Moreover, parasites are prepared without cellular damage whilst maintaining their infectivity.
Purified trypanosomes are required for immunological testing; they are used in the trypanolysis assay, the gold standard in HAT serology. Stained parasites are utilized in the card agglutination test (CATT) for field serology. Antigens from purified trypanosomes, such as variant surface glycoprotein, exoantigens, are also used in various immunoassays. The procedure described here is designed for African trypanosomes; consequently, chromatography conditions have to be adapted to each trypanosome strain, and more generally, to the blood of each species of host mammal.
These fascinating pathogens are easily purified and available to use in biochemical, molecular and cell biology studies including co-culture with host cells to investigate host-parasite relationships at the level of membrane receptors, signaling, and gene expression; drug testing in vitro; investigation of gene deletion, mutation, or overexpression on metabolic processes, cytoskeletal biogenesis and parasite survival.

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers Diethylaminoethyl-cellulose Columns

This method of trypanosome separation from blood depends on their surface charge being less negative than mammalian blood cells. Infected blood is placed and treated on an anion-exchanger column. This method, the most fitting diagnostic for African trypanosomiasis, provides purified parasites for immunological, biological, biochemical, pharmaceutical and molecular biology investigations.

Purificazione dei tripanosomi extracellulare, tra cui africana, dal sangue mediante scambiatori di anioni (colonne Diethylaminoethyl-cellulosa)

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is ...

Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

Fine Needle Aspiration (FNA) is a routine diagnostic procedure essential to both medical and veterinary practices. It consists of the percutaneous aspiration of cells and/or microorganisms from palpable masses, organs or effusions (fluid accumulation in a body cavity) using a thin needle similar to the regular needle used for the venous puncture. The material collected by FNA is in general highly cellular, and the retrieved aspirate is then smeared, air dried, wet-fixed, stained and observed under a microscope. In the clinical context, FNA is an important diagnostic tool that serves as a guide to the appropriate therapeutic management. Because it is simple, fast, minimally invasive and requires limited investment in the laboratory and human resources, it is extensively used by veterinary practitioners, mostly in domestic, but also in farm animals. In studies using animal models, FNA has the advantage that it can be performed repeatedly in the same animal, enabling longitudinal studies through the collection of cells from tumors and organs/tissues over the course of the disease. In addition to routine microscopy, retrieved material can also be used for immunocytochemistry, electron microscopy, biochemical analysis, flow cytometry, molecular biology or in vitro assays. FNA has been used to identify the protozoan parasite Trypanosoma brucei in the gonads of infected mice, opening the possibility for a future diagnosis in cattle.

Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

Fine Needle Aspiration is a technique, whereby cells are obtained from a lesion or organ using a thin needle. Aspirated material is smeared, stained and examined under a microscope for diagnosis or used for molecular biology, cytometry or in vitro analysis. It is cheap, simple, quick and causes minimal trauma.

Rilevamento di parassiti di Trypanosoma Extravascolari da parte dell'aspirazione dell'ago fine

Fine Needle Aspiration (FNA) is a routine diagnostic procedure essential to both medical and veterinary practices. It consists of the percutaneous ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

Misurazione di anticorpi che inducono la fagonenza acquisita naturalmente ai parassiti Plasmodium falciparum mediante un'analisi basata sulla citometria del flusso

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

rilevamento di

Riepilogo

Esplora Altri Video

Preparazione di cellule derivate mieloidi soppressore (MDSC) da Naive e Pancreatiche topi affetti da tumore in citometria a flusso e di smistamento automatico magnetico cellulare Attivato (AutoMACS)

Utilizzo di proteine fluorescenti per monitorare Glycosome Dynamics in Tripanosoma africano

In Vivo Valutazione del roditore Plasmodium parassitemia e merozoite Invasion di Citometria a flusso

Rapida rilevazione omogenea dei saggi biologici Utilizzando Magnetic biopercezione sistema di modulazione

High-throughput di Gene Tagging in

Flusso Virometry per analizzare antigenica spettri di virioni e extracellulare vescicole

Rilevamento e l'arricchimento di Rare antigene-specifiche cellule B per l'analisi del fenotipo e funzione

Purificazione dei tripanosomi extracellulare, tra cui africana, dal sangue mediante scambiatori di anioni (colonne Diethylaminoethyl-cellulosa)

Rilevamento di parassiti di Trypanosoma Extravascolari da parte dell'aspirazione dell'ago fine

Misurazione di anticorpi che inducono la fagonenza acquisita naturalmente ai parassiti Plasmodium falciparum mediante un'analisi basata sulla citometria del flusso

rilevamento di

Riepilogo

Esplora Altri Video

Preparazione di cellule derivate mieloidi soppressore (MDSC) da Naive e Pancreatiche topi affetti da tumore in citometria a flusso e di smistamento automatico magnetico cellulare Attivato (AutoMACS)

Utilizzo di proteine ​​fluorescenti per monitorare Glycosome Dynamics in Tripanosoma africano

In Vivo Valutazione del roditore Plasmodium parassitemia e merozoite Invasion di Citometria a flusso

Rapida rilevazione omogenea dei saggi biologici Utilizzando Magnetic biopercezione sistema di modulazione

High-throughput di Gene Tagging in

Flusso Virometry per analizzare antigenica spettri di virioni e extracellulare vescicole

Rilevamento e l'arricchimento di Rare antigene-specifiche cellule B per l'analisi del fenotipo e funzione

Purificazione dei tripanosomi extracellulare, tra cui africana, dal sangue mediante scambiatori di anioni (colonne Diethylaminoethyl-cellulosa)

Rilevamento di parassiti di Trypanosoma Extravascolari da parte dell'aspirazione dell'ago fine

Misurazione di anticorpi che inducono la fagonenza acquisita naturalmente ai parassiti Plasmodium falciparum mediante un'analisi basata sulla citometria del flusso

Utilizzo di proteine fluorescenti per monitorare Glycosome Dynamics in Tripanosoma africano