Nós gostaríamos de agradecer George Cross para o conselho geral sobre a biologia do tripanossoma. Este trabalho também foi apoiado por uma Fundação Bill e Melinda Gates concessão GCE para DS, a NSF Graduate Research Fellowship (DGE-1325261) para MRM e um NIH / NIAID (concessão # AI085973) para FNP. Agradecemos Galadriel Hovel-Miner para o uso da estirpe contendo o local de gene e reconhecimento I-SCEI.

NOTA: Durante o procedimento, é necessário manter as células no gelo. media frias também devem ser utilizados por toda parte. Colheita 1. Amostra <ol><li> Crescer Lister 427 tripanossomas tensão da corrente sanguínea até uma densidade de 0,5 - 1 milhão / ml. É melhor iniciar culturas com um pequeno número de parasitas. Girar 50 X 10 6 células / amostra durante 10 min a 1500 x g. Certifique-se de deixar 1 x 10 6 células em cultura para mais tarde usar controles de anticorpos como positivos e negativos. NOTA: Este protocolo também pode ser usado em tripanossomas isolados de sangue animal (ver discussão para mais detalhes). </li><li> Pipeta ou deitar fora a maior parte do sobrenadante (deixar cerca de 750 mL). Transferir as células para um tubo de microcentrífuga de 1,5 ml. </li><li> Girar as células a 4 ° C durante 4 minutos a 5.200 xg numa microcentrífuga e remover o sobrenadante. </li></ol> 2. Etiquetagem Magnética <ol><li> Ressuspender as células em 150 ul de meio de cultura (HMI-9 with soro, por exemplo) + anticorpo anti-VSG primário na diluição apropriada. NOTA: O anticorpo anti-VSG é feita em casa e usado numa diluição de 1:50. É importante a utilização de um anticorpo anti-VSG primário que não é marcado com um fluoróforo. </li><li> Vortex células utilizando um vórtice adaptador-60 a uma velocidade de 6 - 8 numa sala fria a 4 ° C durante 10 min. Lavar com 800 - 1.000 l fria HMI-9 com soro. </li><li> Centrifugação a 4 ° C durante 4 min a 5200 xg e o sobrenadante aspirado. Lava-se com 1 ml de HMI-9 com soro e rodar como acima. Aspirar o sobrenadante. Ressuspender o sedimento em 100 ul de HMI-9 com soro. </li><li> Adicionar 110 ul de células magnético ativado triagem microesferas (MSC). Use anti-rato, anti-coelho, ou grânulos anti-biotina, conforme apropriado para o anticorpo anti-VSG primária. Misture bem. células de vórtice em uma sala fria a 4 ° C durante 10 min. </li><li> Enquanto as células são vortex, configure uma separação coluna / amostra magnética ativado em um ímã separação. Certifique-se de ter um receptacle (usamos um tubo de centrífuga de 15 ml) na coluna para recolher o fluxo de passagem. Configurar o aparelho em uma sala fria. </li><li> Adicionar 2 ml de HMI-9 com soro à coluna prime. Após o condicionamento lugar um tubo de centrífuga de 15 ml, sob a coluna para recolha de escoamento a partir de cada amostra. </li><li> Após os 10 minutos de incubação na etapa 2.4, adicione 800-1.000 ul frio HMI-9 com soro. Centrifugação a 4 ° C durante 4 min a 5200 x g e remover o sobrenadante para remover o anticorpo não ligado. Lava-se com 1 ml de HMI-9 com soro e repetir deixando 100 l de sobrenadante. </li><li> Flick o tubo de centrífuga vigorosa e inspeccionar visualmente o tubo a certeza de que o pelete é ressuspenso completamente. Adicionar 1 ml de HMI-9 com soro e pipeta-se para baixo e para certificar-se que as células ressuspensas são bem. </li></ol> 3. Separação Magnética <ol><li> Certifique-se de que as colunas são preparados como descrito acima. Aplicar células de coluna. Recolha de fluxo com células que expressam o VS não-dominanteG. 1 ml de meio + tripanossomas tipicamente leva 6-7 min a fluir através da coluna. </li><li> Lavar 2x com 1 ml de HMI-9 com soro e recolher o fluxo através do mesmo tubo como no passo 3.1. </li><li> Divide escoamento (3 mL) em dois tubos de microcentrifugação de 1,5 ml. NOTA: Estes podem ser combinados mais tarde ou meia pode ser usado para isolar o ARN, etc. </li><li> Remover coluna de ímã. Adicionar 3 ml de HMI-9 com soro à coluna e mergulhar coluna para um tubo de centrífuga de 15 ml para se obter células que expressam a partida, VSG dominante. Retirar 300 ul de material fluido para análise posterior. Manter essas células em gelo. </li><li> Para utilização como um positivo e um controlo negativo, demora aproximadamente 1 milhão de células a partir da cultura inicial de células de controlo em crescimento a 37 ° C (aqueles não esperado para mudar frequentemente) e pipeta-os para um tubo de microcentrífuga de 1,5 ml. NOTA: células de controlo positivo serão coradas com anticorpo marcado com fluoróforo na etapa 4.1. As células de controlo negativo Remana imaculada em todo o resto do protocolo. </li><li> amostras de spin fluxo e amostra de controlo de anticorpos positivo a 4 ° C durante 4 min a 5200 xg e remover o sobrenadante. NOTA: Outra alíquota de 300 ul de células eluídas pode ser utilizado como um controlo positivo para a coloração anti-VSG. </li></ol> 4. A coloração para a identificação de contaminantes e comutadores <ol><li> amostras de fluxo através Ressuspender as células de controlo positivo e em 100 ul de HMI-9 com soro + primário, anti-VSG anticorpo, marcado com fluoróforo na diluição apropriada. NOTA: O anticorpo anti-VSG marcado é feito em casa e usado numa diluição de 1: 1000. A marcado com fluoróforo, anti-VSG pode ser a partir da mesma fonte que o anticorpo não marcado na separação MACS step.There deve ser de 3 ml de amostra por escoamento em dois tubos de microcentrifugação seguintes passo 3.5. </li><li> Para analisar a totalidade do fluxo através de citometria de fluxo, combinar as pelotas em dois tubos de microcentrifugação durante a re-SUSenquanto se aguardam as amostras em 100 l de HMI-9 com anticorpos séricos + anti-VSG. Misture bem. </li><li> células de vórtice em uma sala fria a 4 ° C durante 10 min. Adicionar 800 - 1.000 l fria HMI-9 com soro. amostras de rotação a 4 ° C durante 4 min a 5200 x g e remover o sobrenadante para remover o anticorpo não ligado. </li><li> Lave as células com 1 ml de HMI-9 com soro. amostras de rotação a 4 ° C durante 4 min a 5200 xg e remover o sobrenadante. Durante esta última rotação, girar os-amostras eluídas a partir do passo 3.4 e a amostra de controlo negativo. </li><li> Ressuspender todos escoamento e amostras eluídas em: 148,5 ul HMI-9 com soro, 25 uL grânulos de contagem absoluta e 1,5 ul de solução de iodeto de propídio (175 ul total) coloração. Ressuspender amostras de controlo de anticorpos positivos e negativos em 175 ul HMI-9 com soro. Certifique-se de anotar o número de contas de contagem absolutos / 25 ul para o lote específico utilizado. </li><li> Executar todas as amostras através de um citômetro de fluxo e recolher dados. células mortas da mancha o PI <sup&gt; +. Contaminantes manchar como PI - VSG +. Switchers manchar como PI - VSG - 8. </li><li> Calcular as frequências utilizando citometria de fluxo de dados de comutação. NOTA: Para obter instruções sobre como calcular as frequências de comutação, consulte o arquivo suplementar <a href="https://www.jove.com/files/ftp_upload/54715/Flow_Cytometry_Calculations.docx" target="_blank">Citometria de Fluxo Calculations.docx</a> e Tabela 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.

No que diz respeito à técnica experimental, o componente mais importante do protocolo é manter todas as amostras frias. Tripanossomas internalizar muito rapidamente anticorpo ligado à sua superfície 9, mas este processo é dependente da motilidade, e não influencia o ensaio, enquanto as células são mantidas a 4 ° C. Todas as amostras devem sempre ser mantidos em gelo, e pipetagem deve ser feito rapidamente para minimizar a exposição ao ambiente C laboratório 25 °. Frio HMI-9 com soro deve estar d...

O parasita protozoário, o Trypanosoma brucei, é um agente causador de doenças que afetam os seres humanos (via Trypanosoma brucei gambiense e Trypanosoma brucei rhodesiense) e animais (via Trypanosoma brucei brucei) em toda a África Sub-Sahariana. Ele é transmitido para o hospedeiro mamífero através da saliva do vector mosca tsé-tsé. Ambos Humana e tripanossomíase Africano Animal causar uma carga económica grave em regiões endêmicas, e poucas drogas são disponíveis ou em desenvolvimento para o tratamento de qualquer doença. Compreender mecanismos de evasão imunitária é crítico para o desenvolvimento de drogas para a tripanossomíase. A variação antigênica do revestimento denso, Variante glicoproteína de superfície (VSG) que cobre o parasita é um dos meios primários pelos quais T. brucei evita a resposta do anticorpo de mamífero. Cerca de 2000 variantes do gene VSG existem no genoma tripanossoma, mas só um é transcrito em qualquer momento a partir de uma de ~ 15 expresSites SION. &#39;Comutação&#39; da VSG expresso pode ocorrer quer por cópia de um gene de VSG no local de expressão activa, ou por activação da transcrição de um local de expressão anteriormente silenciosos (revisto em 1). Embora muito trabalho foi dedicado à compreensão variação antigênica em T. brucei, os fatores que influenciam a freqüência de comutação são ainda pouco compreendidos. Estudos até à data têm sido dificultados pelo fato de que, enquanto a comutação ocorre estocasticamente in vitro, freqüências de chaveamento são muito baixos, da ordem de 1 a cerca de 10 6 células 2. Isso torna difícil para medir se um fator dadas aumenta ou diminui frequência de comutação, uma vez que a mudança é difícil de detectar, em primeiro lugar. Antes de 2009, os métodos para isolar switchers em uma dada população foram longo e trabalhoso. Estas células Passaging tripanossomas através de ratinhos imunizados contra o VSG dominante e em seguida colhem incluídosum dias mais tarde três, contando ou centenas de células por imunofluorescência 4,5. Outra estratégia baseia-se na seleção para resistência a drogas para isolar switchers 6. Porque tripanossomas africanos cultivadas in vitro são geralmente composta de uma população grande expressando uma variante importante, e uma população muito menor de switchers que expressam variantes alternativas, referimo-nos à grande variação ao longo deste artigo como o, VSG começando dominante. Ao fazê-lo, nós, de modo algum pretendo afirmar que este importante variante tem maior aptidão do que outras variantes na população. Aqui nós descrevemos um método que pode medir de forma confiável o número de tripanossomas expressando um VSG não dominante numa dada população em 3-4 horas. Este método é particularmente útil para quando se quer determinar se um dado manipulação genética ou tratamento medicamentoso aumenta o número de células ligadas em uma população. Em vez de libertar a população de células que expressam a fazerminant, começando VSG através de drogas ou por meios imunológicos, estas células são eliminadas por primeiro revestindo-os com pérolas magnéticas acopladas a anticorpos contra a VSG dominante e em seguida isolá-los sobre uma coluna magnética. A população ligado é então recolhido no fluxo de passagem e coradas novamente com um anticorpo marcado com fluoróforo anti-VSG para a identificação de contaminantes. A quantificação é conseguida através da adição de um número definido de pérolas de contagem absoluta de cada amostra de modo que a razão de pérolas para células pode ser determinada e utilizada para quantificar o número de comutadores na população 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.

O método aqui descrito foi utilizado para demonstrar que as quebras de cadeia dupla no interior do local de expressão VSG aumentou o número de comutadores numa população 8. Aqui, mostramos resultados representativos de uma população de tripanossomas que têm sido igualmente induzidos para gerar uma ruptura de fita dupla no local expressão. Nós comparar estes tripanossomas para aqueles que não tenham sido induzidas a gerar uma ruptura de fita dupla. A Figura 1...

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.

detecção de<em&gt; Trypanosoma brucei</em&gt; Switching Variant glicoproteína de superfície por Magnetic celular ativado Seleção e citometria de fluxo

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 visualizações.  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.

Vídeo: 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.

Purificação da população de células específicas de separação de células de fluorescência Ativado (FACS)

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

Imunologia e Infecção

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.

Exame da estrutura G-Telomere saliência na 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.

Análise por Citometria de fluxo das células imunológicas Dentro aortas murino

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.

Exame de seleção positiva e negativa Tímico por citometria de fluxo

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.

Detecção de nanopartículas fluorescentes Interações com Primário imunitário subpopulações de células por citometria de fluxo

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.

De alto rendimento Gene Tagging no<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.

Caracterização fenotípica dos macrófagos a partir de rim de rato por Citometria de Fluxo

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.

Classificação de células activadas por fluorescência Para a purificação de células dendríticas plasmocitï¿½des do rato de Medula Óssea

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.

Medição do T celular aloreactividade usando imagens de Citometria de Fluxo

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 cultura In Vitro de macrófagos alveolares murino e humanos

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

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