Estamos em dívida com nossos assistentes técnicos: U. Bröder e H. Shkarlet, que nos ajudou durante procedimentos experimentais. Este trabalho foi parcialmente financiado por subvenções e da UE (contrato n º 601738, UniVax e TRANSVAC2, contraem n º 730964), uma subvenção de Helmholtz-gemeinschaft (HAI-IDR). As fontes de financiamento não influenciou a pesquisa concepção, geração do manuscrito ou decisão de enviá-lo para publicação.

Todos os mouses utilizados neste estudo foram do fundo C57BL/6. Todos os animais foram mantidos em condições isentos de organismos patogénicos. Todos os experimentos foram realizados de acordo com a normativa da lei alemã de proteção animal (TierSchG BGBl. EU S 1105; 25.05.1998) e foram aprovados pela Comissão Baixa Saxônia a ética de experimentos do Animal e do escritório de estado (Baixa Saxônia estado escritório de defesa do consumidor e da segurança alimentar), sob o número de autorização de 33,4-42502-04-13/1281 e 162280.
1. CFSE coloração de OT-eu T células e transferência adoptiva
Nota: OT-os ratos são animais transgenicamente gerados que expressam um receptor de células T (TCR) com α fixo e cadeias β que juntos reconhecem o peptídeo imuno-dominante de óvulos, SIINFEKL10,11. Como resultado, estes ratos têm um número consideravelmente elevado de SIINFEKL específicos CD8+ T células (97%)12 quando comparado com ratos normais ou óvulos vacinados (≤ 1%)13.
<ol><li>Isolamento de CD8 rastreável+ T células de OT-eu camundongos expressando o alelo específico dos linfócitos T Thy1um (Thy1.1):<ol>
<li>Eutanásia em OT 6-9 semanas de idade-eu ratos por inalação de CO2 , seguido por deslocamento cervical14. Dissecar o baço e linfonodos principais (pares inguinais, axilares e cervicais), cortando a pele com uma tesoura cirúrgica, destacando a pele do corpo com a ajuda do alicate cirúrgico e pinça e colocá-los em placas de Petri (15 x 60 mm) cada um contendo um 100 µm poro malha cup para libertação de moagem e células de tecidos.</li>
		<li>Manter os pratos de petri (cada um com uma malha Copa) em 3-5 mL de meio RPMI completo (RPMI 1640, 10% v/v FCS, 100 U/mL penicilina, estreptomicina 50 µ g/mL) mantido no gelo.</li>
		<li>Triture os órgãos com a ajuda de um êmbolo da seringa ou um instrumento similar de estéril (corte prévia não é necessária) e recolher a suspensão única célula resultante em tubos de centrífuga de 15 mL.</li>
		<li>Centrifugar a suspensão de eritrócitos a 300 x g durante 10 minutos a 4 ° C. Desprezar o sobrenadante. Lavar as células em 10 mL de PBS frio por centrifugação e lisar as hemácias do baço, re-suspendendo a pelota em 1 mL/baço do cloreto de amônio buffer (buffer de ACK, comercialmente disponível). Incube as celulas por 1,5 min no gelo e posteriormente lavar as células com 10 mL de PBS frio por centrifugação (como feito antes).</li>
		<li>Ressuspender o sedimento no mesmo tubo em 1 mL de PBS (pH 7,2) contendo 5% fetal soro bovino para isolamento magnético.</li>
	</ol>
</li>
	<li>Para isolamento magnético, proceder à seleção negativa de CD8+ T células usando um kit de isolamento magnético de acordo com as instruções do fabricante ou publicado protocolos15.</li>
	<li>Para executar o CFSE coloração, primeiro contar o número de CD8+ T células obtidas a partir de OT-eu ratos usando um contador de célula automatizada (contador de partículas). Mancha de 1-5 x 107 células/mL em um volume de 1-5 mL com 5 µM CFSE em PBS por 7 min a 37 ° C, protegido da luz em um tubo falcon de 15 ml.</li>
	<li>Saciar a coloração CFSE adicionando o mesmo volume (1:1) de soro fetal bovino para as células e incube-os adicional 7 min a 37 ° C, protegido da luz. As células com 10 mL de PBS lave duas vezes.</li>
	<li>Conte as células usando um contador automatizado de células. Defina o número de células com 3-5 x 107 células/mL de PBS para injetar o 3-5 x 106 células/rato em 100 µ l por via intravenosa (IV) através da veia da cauda.</li>
	<li>Para executar a cauda veia injeções, imobilize os ratos em protecções adequadas. Aquecer a área traseira e a cauda dos ratos deve ser injetado por meio de uma lâmpada de luz vermelha, colocada entre 20 a 25 cm.... os ratos para 1-3 min permitir que a cauda veia vasodilatação. Nota: Isso ajuda a deteção de veia de facilidade e administração da suspensão celular.</li>
	<li>Certifique-se (com a mão colocada entre os ratos e a lâmpada) que não é demasiado quente para os ratos e quando as veias da cauda são claramente visíveis, dirijam-se à injeção. Injete as células na veia de cauda lateral ou dorsal utilizando uma seringa de 1mL com uma agulha de calibre 2516.</li>
</ol>2. imunização (Endo-free OVA + adjuvante)
<ol><li>Coloque os ratos transplantados com OT-eu células (etapa 1.7, Figura 1) em uma câmara de anestesia e administrar isoflurano em oxigênio por uma máquina de anestesia14.</li>
	<li>Quando o mouse está dormindo sob anestesia, tirá-lo da câmara e raspar o pelo do mouse sobre a área sobre o glúteo superficial (laterais, parte inferior das costas), usando uma máquina de corte de cabelo elétrico, a fim de realizar uma injeção limpa com uma boa visão da área de aplicação.</li>
	<li>Injete 50 µ l da vacina, s.c. na área depilada, usando uma agulha de calibre 25.</li>
</ol>3. isolamento de linfócitos e mancha para análise de citometria de fluxo
<ol><li>Isolamento de linfócitos e splenocytes<ol>
<li>Abater os ratos vacinados por inalação de CO2 seguida por deslocamento cervical.</li>
		<li>Extraia o distantes e drenagem dos linfonodos e baço e colocá-los em separado placas de Petri contendo 100 µm poro malha copos para libertação de moagem e células de tecidos. Siga os passos 1.1.2 através de 1.1.3.</li>
		<li>Decante o sobrenadante após centrifugação e ressuspender as pelotas de célula no mesmo volume de remanescente PBS (aprox. 100 µ l).</li>
	</ol>
</li>
	<li>Coloração para análise de citometria de fluxo<ol>
<li>Num tubo de centrífuga de 15 mL prepare uma mistura de mestre que contém os anticorpos coloração nas concentrações descritos na tabela 1, assim, produzindo uma mistura concentrada de coloração de 2x. Prepare-se volume suficiente do mestre mix ter 100 µ l por amostra. Misture as células e a mestre mistura 1:1 e incubar é a 4° C por 30 min. Nota: O volume final de coloração por exemplo deve ser 200 µ l (100 µ l de célula pelota + 100 µ l do mix de mestre de anticorpo).</li>
		<li>Lave as células duas vezes por centrifugação, conforme descrito em 1.1.3, adicionar 10 mL de PBS, duas vezes.</li>
		<li>Ressuspender as células coradas em 0,5-1 mL de PBS para a aquisição e a suspensão de transferência para tubos de citômetro de fluxo. Sempre manter as amostras no gelo e protegido da luz.</li>
	</ol>
</li>
</ol>4. fluxo Cytometry
<ol><li>Sempre pré-filtre as amostras de células usando filtros de 70-100 µm.</li>
	<li>Prepare a coloração única compensação controles para o fluorophores detalhadas na tabela 1 (grânulos ou células). Nota: Compensação deve ser executada no citômetro ou análise software17,18,19 e aplicada a todas as amostras.</li>
	<li>Siga a estratégia associada representada na Figura 2. Brevemente, populações de portão na altura de avançar dispersão vs área de dispersão para a frente (Figura 2A) e novamente dispersão lateral ampla vs área de dispersão lateral (SSA, Figura 2B) a fim de excluir parelhas.</li>
	<li>Portão a população de Figura 2B plotando BV 650 (autofluorescência) vs FITC (CFSE) a fim de discriminar o verdadeiro CFSE manchado células de altas autofluorescente de células. Incluem grânulos ou células coradas com anticorpos conjugados a BV 650 em controles de compensação. Este enredo (Figura 2), da BV 650 vs FITC é usado para portão OT-eu CFSE manchado células.</li>
	<li>Portão da população de C da Figura 2 para a Pe-Cy7 (Thy1.1 - CD90.1-) vs APC (CD8) a fim de distinguir células derivadas de doador vs ratos destinatários. Nota: Células derivadas de OT-eu ratos doadores são Thy1.1+ (CD90.1), (WT C57BL/6, destinatários) derivado de ratos células são Thy1.2+ (CD90.2) (Figura 2D). Alguns laboratórios têm OT-eu ratos em um fundo de Thy1.2 e WT (C57BL/6) em um Thy1.1. Neste caso você tem que usar um anticorpo contra Thy1.2 para OT-eu celular gating.</li>
	<li>Re-portão CD8+ células de Thy1.1+ população plotando-a em um portão constituído por 450 BV (CD4) vs APC (CD8) (Figura 2E).</li>
	<li>Exibir a população fechada na Figura 2E usando um display de histograma do CD8+ células mostrando CFSE (FITC, canal 530/15 - laser azul-), portão a população proliferada, incluindo intensidades de 102 até o nível onde populações a indivisa de controle são (intensidades 10 ~5, dependendo a eficácia do CFSE mancha e a configuração da tensão com o citômetro de fluxo) (Figura 2F).</li>
	<li>Que uma porta adicional (não mostrado na Figura 2) plotagem FITC vs L/D marcador (450/20 canal, laser UV) a fim de avaliar as células mortas, em paralelo à avaliação de proliferação.</li>
	<li>Adquira pelo menos 5000 eventos para os controles de compensação e 10,000 eventos (portão E, Figura 2) das amostras em um citômetro de fluxo. Executar o citômetro de fluxo médio ou baixo (não excedam as taxas de fluxo de 20.000 eventos/s).</li>
</ol>

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Vacinas modernas são idealmente composedof antígeno purificado e adjuvantes, com a possível adição de um sistema de entrega como lipossomas, vírus-como partículas, nanopartículas ou vetores ao vivo. Um aspecto fundamental durante a criação de uma vacina é escolher o certo adjuvante de acordo com as necessidades clínicas. Parte do escopo poderia envolver favorecendo um humoral vs. resposta imune celular (ou ambos), a eleição de um local contra uma resposta imune sistêmica (ou ambos) e o tipo de memória que...

Linfócitos T citotóxicos (CTL) induzindo vacinas são principais intervenções terapêuticas que foram desenvolvidas para lutar contra certos tipos de câncer1. CTL são também importantes para vacinas profiláticas contra patógenos intracelulares2. Além disso, o CTL são dentre os mecanismos de defesa imune alguns funcionalmente ativos em populações de risco tais como neonatos3,4 , quem também dependem de CTL para combate de infecções início vida5. A este respeito, as vacinas contra o vírus sincicial respiratório (VSR) que foram desenvolvidas com um adjuvante que não eliciam respostas CTL (alume) resultaram em um completo fracasso da vacina levando a sérias complicações com infecções em lactentes de6. Estes efeitos negativos da vacinação podem ser revertidos por um CD8+ de resposta de células T7. Nós demonstramos anteriormente que as principais citocinas (interferons do tipo I) eliciadas algum estimulador de agonistas de genes (picada) de interferon são essenciais para as respostas CTL geradas por estes adjuvantes8, em parte, medindo-se a proliferação de OT-I T pilhas após vacinação e usando estes resultados como uma medida de CTL induzindo capacidades observadas em estendem de horários de vacinação9. A medição da proliferação de OT-eu CD8+ T em um rato de destinatário tipo selvagem (WT) C57BL/6 células por carboxyfluorescein grufo tintura de éster (CFSE) diluição é uma robusta estimativa da capacidade do adjuvante de uma vacina para gerar Cruz-ferragem de SIINFEKL, (o peptídeo imuno-dominante da ovalbumina, óvulos). Variações desta técnica são amplamente utilizadas para a avaliação da proliferação de OT-eu CD8+ e OT-II CD4+ T células. Por exemplo, ela tem sido usada na ausência de citocinas selecionadas (ratos KO) ou para medir a eficácia da vacina após retirada de antigénios em animais WT. Criámos um protocolo curto (4 dias experimento) em que após a transferência passiva de OT CFSE-manchado-eu CD8+ T cells, uma imunização (s.c.) subcutânea, consistindo de uma dose de 50 µ g de OVA livre de endotoxinas suplementado com adjuvantes de teste é administrado (Figura 1). O acompanhamento dos resultados 48 h após a vacinação fornece uma confiável prova da capacidade do adjuvante para gerar respostas CTL. Por esta estratégia, é possível avaliar a potência da resposta imune local no nó de linfa drena após a imunização, bem como a extensão da resposta, medindo-se a atividade CTL no baço (ou distante dos gânglios linfáticos).

<table>
	<tbody>
		<tr>
			<td>BD LSR Fortessa Cell Analyzer</td>
			<td>BD</td>
			<td>Special Order</td>
			<td>Flow Cytometer</td>
		</tr>
		<tr>
			<td>CFSE</td>
			<td>Molecular Probes</td>
			<td>C34554</td>
			<td>Proliferation Dye</td>
		</tr>
		<tr>
			<td>MojoSort Mouse CD8 T Cell Isolation Kit</td>
			<td>Biolegend</td>
			<td>480007</td>
			<td>Magnetic Isolation Beads and antibodies for negative selection of untouched CD8 T cells.</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation</td>
			<td>Molecular Probes</td>
			<td>L23105</td>
			<td>Dead Cell Marker</td>
		</tr>
		<tr>
			<td>CD90.1 (Thy-1.1) Monoclonal Antibody (HIS51), PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-0900-82</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>APC anti-mouse CD8a Antibody</td>
			<td>BioLegend</td>
			<td>100712</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>BV421 Rat Anti-Mouse CD4</td>
			<td>BD</td>
			<td>740007</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>Z2 coulter Particle count and Size Analyzer</td>
			<td>Beckman Coulter</td>
			<td>9914591DA</td>
			<td>Cell counter. Z2 Automated particle/cell counter</td>
		</tr>
		<tr>
			<td>EndoGrade Ovalbumin (10 mg)</td>
			<td>Hyglos(Germany)</td>
			<td>321000</td>
			<td>Ovalbumin endotoxin free tested.</td>
		</tr>
		<tr>
			<td>Cell Strainer 100&#181;m nylon</td>
			<td>Corning</td>
			<td>352360</td>
			<td>Cell strainer (100 &#181;m pore mesh cups).</td>
		</tr>
		<tr>
			<td>Sample Vials</td>
			<td>Beckman Coulter</td>
			<td>899366014</td>
			<td>Sample vials for Z2 automated counter</td>
		</tr>
		<tr>
			<td>C57BL/6 mice (CD90.2)</td>
			<td>Harlan (Rossdorf, Germany)</td>
			<td></td>
			<td>Company is now Envigo</td>
		</tr>
		<tr>
			<td>OT-I (C57BL/6 background, CD90.1)</td>
			<td>Harlan (Rossdorf, Germany)</td>
			<td></td>
			<td>Inbreed at our animal facility. Company from where adquired is now Envigo</td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Fischer (Corning)</td>
			<td>14-959-5</td>
			<td>Corning Falcon Round-Bottom Polystyrene Tubes</td>
		</tr>
		<tr>
			<td>Falcon 15 mL tubes</td>
			<td>Fischer (Corning)</td>
			<td>05-527-90</td>
			<td>Falcon 15mL Conical Centrifuge Tubes</td>
		</tr>
		<tr>
			<td>PBS (500 mL)</td>
			<td>Fischer (Gibco)</td>
			<td>20-012-027</td>
			<td>Gibco PBS (Phosphate Buffered Saline), pH 7.2</td>
		</tr>
		<tr>
			<td>Red lamp (heating lamp)</td>
			<td>Dirk Rossmann GmbH (Germany)</td>
			<td>405096</td>
			<td>Heating infrred lamp (100 wats)</td>
		</tr>
		<tr>
			<td>IsoFlo (Isoflurane)</td>
			<td>Abbott Laboratories (USA)</td>
			<td>5260.04-05.</td>
			<td>Isoflurane anesthesic (250 mL flask).</td>
		</tr>
		<tr>
			<td>Tabletop Anesthesia Machine/Mobile Anesthesia Machine with CO2 Absorber</td>
			<td>Parkland Scientific</td>
			<td>V3000PK</td>
			<td>Isoflurane anesthesia machine.</td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>11-875-093</td>
			<td>Base medium with Glutamine (500 mL)</td>
		</tr>
		<tr>
			<td>Pen-Strept antibiotic solution (Gibco)</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>15-140-148</td>
			<td>Gibco Penicillin-Streptomycin (10,000 U/mL)</td>
		</tr>
		<tr>
			<td>Fetal Bobine Serum (Gibco)</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>10082147</td>
			<td>Fetal Bovine Serum, certified, heat inactivated, US origin</td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer (100 ml)</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>A1049201</td>
			<td>Amonium Chloride Potasium (ACK) Whole Blood Lysis Buffer, suitable for erytrocyte lysis in spleen suspensions also</td>
		</tr>
		<tr>
			<td>Plastic Petri Dishes</td>
			<td>Nunc (distributed by ThermoFischer)</td>
			<td>150340</td>
			<td>60 x 15mm Plastic Petri Dish, Non-treated</td>
		</tr>
		<tr>
			<td>Cell Clump Filter</td>
			<td>CellTrics (Sysmex)</td>
			<td>04-004-2317</td>
			<td>CellTrics&#174; 50 &#956;m, sterile</td>
		</tr>
	</tbody>
</table>

rapid in vivo assessment of adjuvant's cytotoxic t lymphocytes generation capabilities for vaccine development

A avaliação de vacinas de subunidade moderno revela que a geração de anticorpos neutralizantes é importante mas não suficiente para a seleção de adjuvante. Portanto, adjuvantes com capacidades imuno-estimulatórios humorais e celulares que são capazes de promover respostas de linfócitos (CTL) T citotóxicas são urgentemente necessárias. Assim, fiel, monitoramento de adjuvante que induzir Cruz-ferragem e posteriormente aumentar a geração de CTL representa um passo crucial no desenvolvimento de vacinas. Aqui apresentamos um pedido de um método que usa SIINFEKL específicos (OT-eu) células T para monitorar a Cruz-apresentação do modelo do antígeno ovalbumina (OVA) na vivo na presença de candidatos de diferentes adjuvantes. Este método representa um teste rápido para selecionar adjuvantes com os melhores recursos de cruz-ferragem. A proliferação de CD8+ T células é a indicação mais valiosa da Cruz-ferragem e também é considerado como um correlato de cruz-apresentação induzida por adjuvante. Esta característica pode ser avaliada em diferentes órgãos imunes como linfonodos e baço. A extensão da geração CTL também pode ser monitorizada, conferindo-lhe insights sobre a natureza de um local (drenagem do linfonodo principalmente) ou uma resposta sistêmica (distante dos gânglios linfáticos e/ou baço). Ainda mais esta técnica permite várias modificações para testes de drogas que podem inibir a percursos específicos da Cruz-apresentação e também oferece a possibilidade de ser usado em diferentes cepas de ratos geneticamente modificados e convencionais. Em resumo, o aplicativo que apresentamos aqui será útil para laboratórios de vacina em indústria ou do meio académico que desenvolvem ou modificar adjuvantes químicos para pesquisa de vacina e desenvolvimento.

Para testar os tratamentos utilizando uma combinação diferente de adjuvantes (ADJ1 e ADJ2), avaliamos a capacidade de geração de CTL medindo-se a proliferação de OT enviaram transferido-eu CD8+ T células por citometria de fluxo (Figura 2). Por isso, nós anteriormente manchadas de células isoladas da drenagem dos gânglios linfáticos e baço (tabela 1). Medindo-se a proliferação de CD8+ T células nos linfonod...

Watch this Scientific Journal Video about Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development at JoVE.com

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

Apresentamos aqui um pedido de uma norma técnica imunológica (CFSE manchado OT-eu proliferação) destinados a monitorar rapidamente mediada por adjuvante T linfócitos citotóxicos (CTL) geração in vivo. Esta estimativa rápida das capacidades CTL é útil para o desenvolvimento de vacinas profiláticas contra patógenos intracelulares, bem como as vacinas terapêuticas do câncer.

Rápida na Vivo avaliação dos recursos de geração de linfócitos de T citotóxicos do adjuvante para desenvolvimento de vacinas

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

rapid-vivo-assessment-adjuvant-s-cytotoxic-t-lymphocytes-generation

Research

JoVE Journal

Medicine

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

8.6K visualizações.  Helmholtz Centre for Infection Research. Apresentamos aqui um pedido de uma norma técnica imunológica (CFSE manchado OT-eu proliferação) destinados a monitorar rapidamente mediada por adjuvante T linfócitos citotóxicos (CTL) geração in vivo. Esta estimativa rápida das capacidades CTL é útil para o desenvolvimento de vacinas profiláticas contra patógenos intracelulares, bem como as vacinas terapêuticas do câncer.

Vídeo: Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

Teratoma Generation in the Testis Capsule

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers. This makes them a potentially valuable tool for the treatment of many different diseases. With the advent of induced pluripotent stem cells (iPSCs) and continuing research with human embryonic stem cells (hESCs) there is a need for assays that can demonstrate that a particular cell line is pluripotent. Germline transmission has been the gold standard for demonstrating the pluripotence of mouse embryonic stem cell (mESC) lines1,2,3. Using this assay, researchers can show that a mESC line can make all cell types in the embryo including germ cells4. With the generation of human ESC lines5,6, the appropriate assay to prove pluripotence of these cells was unclear since human ESCs cannot be tested for germline transmission. As a surrogate, the teratoma assay is currently used to demonstrate the pluripotency of human pluripotent stem cells (hPSCs)7,8,9. Though this assay has recently come under scrutiny and new technologies are being actively explored, the teratoma assay is the current gold standard7. In this assay, the cells in question are injected into an immune compromised mouse. If the cells are pluripotent, a teratoma will eventually develop and sections of the tumor will show tissues from all 3 germ layers10. In the teratoma assay, hPSCs can be injected into different areas of the mouse. The most common injection sites include the testis capsule, the kidney capsule, the liver; or into the leg either subcutaneously or intramuscularly11. Here we describe a robust protocol for the generation of teratomas from hPSCs using the testis capsule as the site for tumor growth.

Human pluripotent stem cells (hPSCs) have the potential to treat a myriad of different diseases. The utility of these cells lies in the fact that they can differentiate into any cell type in the body. Here we describe the teratoma assay, which is used to demonstrate the pluripotence of hPSCs.

Geração Teratoma na Cápsula do Testículo

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers.  This makes them a ...

Medicina

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

Ensaio de Point-of-Care rápida de eficácia anticoagulante enoxaparina em sangue total

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19 F MRI para Rastreamento celular

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

Citotóxicos Eficácia da Terapia Fotodinâmica no osteossarcoma Células<em&gt; In Vitro</em

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Seqüente<em&gt; In vivo</em&gt; Imagem de osteogênicas células-tronco / progenitoras durante o reparo de fratura

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Avaliação do cancro do ovário esferóide Apego e invasão de células mesoteliais em Tempo real

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

Coleta e Extração de DNA de saliva para Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Avaliação da viabilidade de gordura humana injecção em ratinhos nu com Micro-Tomografia Computadorizada

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Geração de Microtumors Usando 3D Biogel Humano Sistema de Cultura e células de glioblastoma derivado do paciente para Kinomic Profiling e Testes resposta à droga

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

Depleção de células do rato de xenoenxertos de tumor humano Melhora Significativamente Análise Downstream de células alvo

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...