Este trabalho foi financiado por doações fromthe Fundo Belga de Pesquisa Científica (FNRS), Les Amis de l&#39;Institut Bordet, FNRS-operação Télévie, Câncer da Bélgica Plan, Fonds Lambeau-Marteaux, Fonds JC Heuson e Fonds Barsy.

NOTA: Todos os espécimes foram adquiridos através de um protocolo aprovado pelo Comitê de Ética Médica do Instituto Jules Bordet com consentimento informado por escrito obtido de cada paciente. 1. Preparação do homogenato de tecido <ol><li> Dissecar tecidos ressecados (tecido maligno e normal ressecado da sala de cirurgia) estão no laboratório de patologia por pessoal treinado para a coleta imediata. Tumor, NANT (tomado a maior distância possível do tumor) e fragmentos de tecidos normais são rotineiramente processados ​​dentro de 1-3 horas de excisão cirúrgica em um laboratório BSL2 utilizando procedimentos de biossegurança padrão para os tecidos humanos. Um diagrama de fluxo do protocolo é ilustrada na Figura 1. </li><li> Pesar todos os fragmentos de tecido (normal, NANT e tumorais) e medir o comprimento, largura e altura (comprimento x largura x altura). Este é um passo importante para a posterior normalização de subpopulações de células, RNA extraído, etc. NOTE: A gama de tamanho da amostra é de 100 a 10.000 mm3, sem gordura, se possível. </li><li> Imprimir o fragmento de tumor numa lâmina de vidro para H &amp; E coloração para verificar se o tecido é realmente parte do tumor. <ol><li> Faça isso pressionando uma lâmina de microscópio de vidro sobre o fragmento de tumor e aplicar uma leve pressão com os dedos por alguns segundos. </li><li> Corrigir o diapositivo com isopropanol durante 2 min, seguido por um passo de lavagem em água. Contracorante o tecido durante 30 segundos com hematoxilina de Mayer. </li><li> Lave o slide em seis banhos de água. Mancha em Floxina B 2% por 15 segundos. </li><li> Lave em um banho de água, seguido de quatro banhos de isopropanol e terminar com um banho de água. </li><li> Incubar em isopropanol, durante 1 min e de drenagem. Limpar em dois banhos de xileno. </li><li> Montar com meio de montagem com base xileno. Examinar para a celularidade do tumor (Figura 2). NOTA: Principalmente, as células tumorais ficar com o slide impressa - o estroma, linfóide ou adipose células permanecem raramente, deixando espaços entre as células de tumor (Figura 2). </li></ol></li><li> Inserir o fragmento de tecido em um pequeno prato de cultura contendo 1 mL de o, meio de células hematopoiéticas isento de soro quimicamente definido (daqui em diante referido como meio) à temperatura ambiente e corte em pedaços pequenos (~ 1 - 2 mm 2), utilizando um filtro estéril bisturi. </li><li> Transfira tudo (fragmentos de tecido + média) para um tubo de Dissociator C mecânica. </li><li> Lavar a placa de Petri e bisturi com 2 ml de meio com uma pipeta de Pasteur e adicionar este ao tubo C (volume de meio de dissociação = 3 ml). </li><li> Use a A.01 programa Dissociator mecânica para tubos C (o programa mais suave) para homogeneizar os fragmentos de tecido em uma suspensão de células individuais. Colocar o tubo C no aparelho e executa o programa de duas vezes em sucessão (um ciclo = 25 seg). NOTA: Este procedimento homogeneização foi estabelecido e validado para o tecido mamário humano, outro tumou ou tecido tipos pode precisar usar um programa diferente e deve ser testado pela primeira vez. </li><li> Remover o tubo C do aparelho e decanta-se o homogeneizado directamente para um filtro de células de 40 mm assentado em um tubo de 50 ml. Usando a mesma pipeta de Pasteur, como na etapa 1.6, transferir qualquer líquido remanescente no tubo C para o coador de células. </li><li> Transferir o líquido filtrado para um tubo de 15 ml utilizando uma ponta de micropipeta 1 ml. Temporariamente manter o filtro de células e o seu tubo de 50 ml. </li><li> Lavar o tubo C com um adicional de 3 ml de meio e transferir este, de novo usando a mesma pipeta de Pasteur, como na etapa 1.6, para o filtro de células ainda sentado no tubo de 50 ml. Esprema um montante máximo do líquido residual preso no tecido unhomogenized para dentro do tubo de 50 ml, movendo-a suavemente ao redor do filtro com uma pipeta Pasteur limpa ou 1 ml dica que é posteriormente jogado fora para evitar a contaminação do fluido. </li><li> Coloque o filtro de células de cabeça para baixo em tele tubo original C e enxagua-se com 3 ml de meio de modo que o tecido unhomogenized cai de volta para dentro do tubo C. </li><li> Re-homogeneizar como no passo 1.7 para dois ciclos do programa A.01. </li><li> Coloque esta segunda homogenato através do filtro de células sentado no tubo de 50 mL, enxaguar o tubo C de novo com 3 mL de meio (tal como no passo 1.10) e de transferência com a pipeta de Pasteur para o coador de células sentado no tubo de 50 ml novo apertando um máximo quantidade de líquido a partir do tecido conjuntivo residual preso no filtro celular. </li><li> Neste ponto, um volume de ~ 2,5 ml é no tubo de 15 ml e ~ 9 ml no tubo de 50 ml. </li></ol> 2. Separação do sobrenadante de células e tecidos <ol><li> Centrifugar os homogeneizados em 15 mL e tubos de 50 ml durante 15 minutos a 600 xg à temperatura ambiente. </li><li> Decantar o SN do tubo de 15 ml para um tubo limpo e armazenar temporariamente a 4 ° C. Este sobrenadante = tumor primário, NANT ou ti normaisssue SN (volume final de 2,5 ml) é posteriormente clarificada e aliquotadas antes do armazenamento a -80 ° C para futuras análises (ver abaixo). </li><li> Descartar o sobrenadante do tubo de 50 ml. </li><li> Suavemente ressuspender ambos os peletes de células, num volume final de 1 ml de meio. Resumidamente, em primeiro lugar quebrar suavemente o sedimento de células em ambos os tubos (tocando o tubo sobre uma superfície dura). Ressuspender o sedimento de células soltas no 50 ml com 500 ul de meio e transferir esta suspensão celular para o tubo de 15 ml para ressuspender o sedimento de segundo. Repetir esta etapa uma vez com o segundo 500 ul de meio para a recuperação máxima de células. </li><li> Transferir 10 uL da suspensão de células para um pequeno tubo, misturar com 10 ul de azul de tripano (diluição 1: 1) e contar o número de células viáveis ​​utilizando um hemocitómetro. NOTA: Neste ponto, uma fracção da suspensão de células podem também ser analisados ​​por citometria de fluxo para avaliar o tamanho das células, granularidade e se desejado um número limitado de marcadores de subpopulação para mais precise avaliação da distribuição de célula relativa no homogeneizado antes da análise extensiva ou experimentação. Todas as análises por citometria de fluxo incorporar rotulagem CD45 para normalizar as subpopulações. </li><li> Agregar as células por centrifugação a 300 xg durante 10 min à temperatura ambiente. As células do tumor, NANT, ou tecido normal são agora pronta para purificação adicional ou análise. Estes passos adicionais são melhor quando realizada no mesmo dia que a cirurgia. NOTA: Para a análise de citometria de fluxo, mas não de separação de células dos glóbulos vermelhos residuais devem ser lisadas após marcação com anticorpos pela adição de 0,4 ml de tampão de lise de glóbulos vermelhos para a pelete de células, imediatamente vortex durante 1 seg e incubar um mínimo de 10 min a sala temperatura (protegido da luz), antes da análise. </li></ol> 3. Clarificação do sobrenadante Tissue <ol><li> Centrifugar os tubos de 1,5 ml com SN tecido a 15.000 xg durante 15 min a 4 ° C. </li><li> Retire com cuidado tele sobrenadante sem tocar ou perturbar o sedimento. Transferir para um tubo limpo (ou tubos), dependendo do número e volume de alíquotas desejados. </li><li> Armazenar o sobrenadante a -80 ° C para uso futuro. </li></ol> 4. O sangue do paciente <ol><li> Recolher uma amostra de sangue de cada paciente como um controlo por venipunctura para dentro de tubos heparinizados o dia antes da cirurgia. Centrifuga-se o sangue a 400 xg durante 10 min a 20 ºC, com o travão para se obter o plasma e um creme leucocitário. </li><li> Remover o plasma e esclarecer-o por centrifugação a 10.000 xg durante 15 min a 20 ºC. Alíquotas e armazenamento de plasma a -80 ° C para uso futuro. Diluir o creme leucocitário em meio </li><li> Separam-se as células mononucleares utilizando centrifugação em gradiente de padrão de Ficoll-Hypaque antes da análise imediata, isolamento subpopulação, / ARN de extracção de ADN / proteína ou de criopreservação. </li></ol> 5. Citometria de Fluxo <ol><li> Células etiqueta de acordo com a produçãoinstruções de r, a 4 ° C, protegidos da luz. Lisar as células vermelhas do sangue em suspensões de células a partir de fragmentos de tecido após a marcação de anticorpos por adição de 0,4 ml de tampão de lise de células para a pelete de células. </li><li> Vortex imediatamente por 1 segundo e incubar um mínimo de 10 min à temperatura ambiente, protegida da luz. Passe as células marcadas em um citômetro de fluxo para aquisição de dados, sem lavar. </li></ol>

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The authors declare that no conflict of interest exists.

Este estudo descreve um método otimizado para o preparo rápido de homogeneizados normais e malignas do tecido mamário, sem digestão enzimática para classificação subseqüente celular, extração, criopreservação e / ou análise fenotípica de CD45 + subpopulações. O objetivo dessa abordagem experimental é produzir imagens da TIL que refletem de perto seu estado in vivo e compará-los aos tecidos normais com mínima manipulação dos tecidos frescos da sala de cirurgia. Até o momento, o n...

The tumor microenvironment is composed of various cell types with numerous studies showing they each play distinct and important roles in tumorigenesis1,2. These include, but are not limited to, infiltrating immune cells, stromal cells, endothelial cells and tumor cells3. Ex vivo studies of tumor infiltrating lymphocytes (TIL; CD45+ cells or leukocytes, which are predominantly lymphocytes in breast tumors) from fresh human tissue samples is made difficult by their low frequency, the small sample sizes often available for research and the potential for loss of viability during extraction. Because immune cells infiltrating tumors are usually present as passengers rather than permanent residents in general they are easier to release from the tissue matrix.
Dissociating tumor tissue while maintaining cellular integrity is technically challenging and has traditionally been performed using a combination of mechanical and enzymatic steps to prepare single cell suspensions4-8. This approach involves lengthy incubation periods and is associated with a significant reduction in cell viability as well as the loss of cell surface receptors by enzymatic cleavage. High quality flow cytometric studies characterizing TIL in the tumor microenvironment as well as clean purifications of CD45+ subpopulations by flow cytometry or antibody-coated beads are more difficult to achieve from enzyme-digested tumor tissue. In addition, the supernatant (SN) from the resulting tumor homogenate is not amenable to further analysis including quantification of secreted proteins (cytokines, chemokines, immunoglobulins or tumor antigens) or experimental treatment of normal cells, because of the potential for protein degradation in the enzymatic digests.
In our search for a method to prepare single cell homogenates from breast tissues [including tumor, non-adjacent non-tumor (NANT) and normal (from mammary reductions) breast tissues] without enzymatic digestion, we tested a variety of mechanical homogenization techniques. Homogenates prepared using a mechanical dissociator had increased cell viability (2-fold) and total cell recovery (2-fold) while preserving surface receptor expression. Enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells suggesting they were all released in the initial homogenate. Thus, this rapid and simple approach allows both qualitative and quantitative assessment of the CD45+ subpopulations present in various normal and malignant human tissues. An added advantage of this approach is that the SN from the initial homogenate (primary tissue SN) can be collected and stored for further analysis or experimentation.

<table border="0" cellpadding="0" cellspacing="0" width="980">
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;">Equipment</td>
			<td style="width:163px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:409px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">GentleMacs Dissociator&#160;</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-093-235</td>
			<td>BD Medimachine is somewhat equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Centrifuge 5810 R</td>
			<td style="width:163px;">Eppendorf</td>
			<td style="width:137px;">N/A</td>
			<td>or other standard table top centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Centrifuge 5417 R</td>
			<td style="width:163px;">Eppendorf</td>
			<td style="width:137px;">N/A</td>
			<td>or other standard microcentrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Esco Class II A2 Biosafety Cabinet</td>
			<td>ESCO global</td>
			<td style="width:137px;">N/A</td>
			<td>or other standard BSL2 hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Inverted Microscope</td>
			<td style="width:163px;">Nikon eclipse TS100</td>
			<td style="width:137px;">N/A</td>
			<td>or other microscope compatible for a hemacytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">B&#252;rker Chamber</td>
			<td style="width:163px;">Marienfield&#160;</td>
			<td style="width:137px;">640210</td>
			<td>or other standard hemacytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Navios Flow Cytometer</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:137px;">N/A</td>
			<td>or other flow cytometer (8-10 color recommended)</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;"></td>
			<td style="width:163px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;">Materials</td>
			<td style="width:163px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">GentleMacs C-Tube</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-096-344</td>
			<td>BD Medimachine uses Filcon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Cell Culture Dish</td>
			<td style="width:163px;">Sarstedt</td>
			<td style="width:137px;">72,710</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Disposable Scalpel</td>
			<td style="width:163px;">Swann-Morton</td>
			<td style="width:137px;">0510</td>
			<td>or standard single use sterile scalpel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD Cell Strainer 40&#181;m</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">734-0002</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD Falcon Tube 50mL</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">352070</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD Falcon Tube 15mL</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">352097</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD FACS Tube 5mL</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">352008</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Sterile Pasteur Pipette 5 mL&#160;</td>
			<td style="width:163px;">VWR</td>
			<td style="width:137px;">612-1685</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Microfuge Tube 1.5 mL</td>
			<td style="width:163px;">Eppendorf</td>
			<td style="width:137px;">7805-00</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;"></td>
			<td style="width:163px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;">Reagents</td>
			<td style="width:163px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">X-Vivo 20</td>
			<td style="width:163px;">Lonza</td>
			<td style="width:137px;">BE04-448Q</td>
			<td>serum-free medium recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Phosphate buffered saline</td>
			<td style="width:163px;">Lonza</td>
			<td style="width:137px;">BE17-516F</td>
			<td>standard physiological PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Trypan blue&#160;</td>
			<td style="width:163px;">VWR</td>
			<td style="width:137px;">17942E</td>
			<td>or other vital stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">VersaLyse</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:137px;">A09777</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Fixable viability Dye eFluor 780&#160;</td>
			<td style="width:163px;">eBioscience</td>
			<td style="width:137px;">65-0865-14</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD3 FITC</td>
			<td style="width:163px;">BD Biosciences</td>
			<td style="width:137px;">345763</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD3 Vio Blue</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-094-363</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD4 PE</td>
			<td style="width:163px;">BD Biosciences</td>
			<td style="width:137px;">345769</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD4 APC</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-091-232</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD8 ECD</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:137px;">737659</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD8 PerCP</td>
			<td style="width:163px;">BD Biosciences</td>
			<td style="width:137px;">345774</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD19 APC-Vio770</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-096-643</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD45 VioGreen</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-096-906</td>
			<td>for flow cytometry experiments</td>
		</tr>
	</tbody>
</table>

a simple and rapid protocol to non-enzymatically dissociate fresh human tissues for the analysis of infiltrating lymphocytes

A capacidade das células malignas para evadir o sistema imune, caracterizado por escape do tumor a partir de ambas as respostas imunes inatas e adaptativas, é agora aceite como uma característica importante do cancro. Nossa pesquisa sobre câncer de mama centra-se no papel ativo que os linfócitos tumor infiltrando desempenhar na progressão do tumor e evolução de pacientes. Com esse objetivo, foi desenvolvida uma metodologia para o rápido isolamento de células linfóides intactas de tecidos normais e anormais em um esforço para avaliá-los próximo ao seu estado natal. Homogeneizados preparados utilizando um show Dissociator mecânica tanto maior recuperação e viabilidade celular, preservando a expressão do receptor de superfície em comparação tecidos para enzima-digerida. Além disso, a digestão enzimática do material insolúvel restante não recuperou células adicionais CD45 +, indicando que as medidas quantitativas e qualitativas no homogeneizado primária provável genuinamente refletem infiltrando as subpopulações no fragm tecidoent. As células linfóides em homogenatos estes podem ser facilmente caracterizadas utilizando imunológica (fenótipo, proliferação, etc.) ou molecular (DNA, RNA e / ou proteína) se aproxima. As células CD45 + pode também ser utilizado para a purificação subpopulação, expansão in vitro ou de criopreservação. Um benefício adicional desta abordagem é que o sobrenadante de tecido primário a partir dos homogeneizados podem ser utilizados para caracterizar e comparar as citocinas, quimiocinas e imunoglobulinas, antigénios presentes em tecidos normais e malignos. Este funções de protocolo extremamente bem para tecidos mamários humanos e deve ser aplicável a uma grande variedade de tecidos normais e anormais.

A digestão enzimática de fragmentos de tecido, quer com soluções comercialmente disponíveis de dissociação de tecido ou várias misturas de laboratório de colagenase, ADNase e / ou inibidores de hialuronidase, clivar uma grande variedade de receptores na superfície das células. Os nossos estudos, inicialmente centrado sobre as células T CD4 + que infiltram os tumores de mama, foram rapidamente apresentados com um grande problema técnico, devido à clivagem dos receptores de CD4 de superfície util...

Watch this Scientific Journal Video about A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes at JoVE.com

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

Um protocolo simples e rápido para não enzimática Separar frescos Tecidos Humanos para a Análise de infiltrar Linfócitos

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

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Research

JoVE Journal

Immunology and Infection

19.9K visualizações.  Université Libre de Bruxelles. This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

Vídeo: A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Combinação de adesivos de fita baseada em amostragem e de fluorescência In situ Hibridação para detecção rápida de Salmonella On Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Imunologia e Infecção

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

Um Protocolo de Chelex simples para extração de DNA de<em&gt; Anopheles</em&gt; Spp.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

Um protocolo simples para agregação plaquetária mediada por<em&gt; Plasmodium falciparum</emEritrócitos&gt; infectados em uma configuração com poucos recursos

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Rápida análise de aberrações cromossômicas em linfócitos B mouse por PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Protocolo de isolamento eficiente para linfócitos B e T a partir de tonsilas palatinas Humanos

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

Uma abordagem automatizada-Semi Preparando Antibody Cocktails para Análise imunofenotípica de Sangue Periférico Humano

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Um método rápido para processamento de sangue para aumentar o rendimento dos níveis plasmáticos de peptídeos no sangue humano

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Uma abordagem simples e eficiente para construir vectores de vírus mutante Vaccinia

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

Um protocolo otimizado para analisar glicólise e respiração mitocondrial nos linfócitos

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

Quantificação rápida de blastogénese induzida por Mitogénio em linfócitos T para identificar drogas imunomoduladoras

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...