Este trabalho é apoiado pelo NIH conceder R00 CA160574 (SFB) e por uma bolsa do Programa de Formação Biotecnologia Rutgers (a SMM).

Este procedimento foi aprovado pelo Comitê de Cuidados e Uso Animal Institucional da Universidade Rutgers, a Universidade Estadual de Nova Jersey. Ratos foram tratados de acordo com o Guia do NIH para o Cuidado e Uso de Animais de Laboratório. Os cientistas devem consultar as suas organizações nacionais de animais, e institucionais para as diretrizes estabelecidas e aprovadas. Antes de começar, preparar as soluções listadas na Tabela 1. 1. B Isolamento e Ativação Celular Eutanásia um rato usando CO 2, seguida por deslocação cervical. Posicione o mouse para o lado esquerdo do corpo é para cima e pulverizar o mouse com etanol a 70% até a pele está úmida. Dissecar o baço e coloque em um 35 milímetros de cultura de tecidos prato com tampão de lavagem 2 ml. Em uma câmara de fluxo laminar, quebrar-se suavemente o baço em placas de cultura de tecidos utilizando a extremidade plana de uma seringa de 5 ml. <ol><li> Insira uma malha de nylon 70 um em um 50tubo ml e transferir a amostra de baço em 2 ml de tampão de lavagem para a malha de nylon. Suavemente perturbar o baço na malha usando a extremidade plana da seringa. </li><li> Lavar a parte de trás da seringa e a placa 35 mm, com 8 ml de tampão de lavagem e filtrar através de malha de nylon de 70 ^ m. Centrifugar a 300 xg durante 10 min a 4 ° C. </li><li> Aspirar e desprezar o sobrenadante e ressuspender o sedimento em 3 ml de tampão de lise ACK. Pipeta-se para baixo e para interromper momentaneamente aglomerados e incubar durante 5 min. Adicionar 10 ml de tampão de lavagem e centrifugar a 300 xg durante 10 min a 4 ° C. </li><li> Aspirar e desprezar o sobrenadante e ressuspender o pellet tocando no fundo do tubo, em seguida por pipetagem em 1 ml de tampão de lavagem. Adicionar 50 ul de microesferas anti-CD43 MACS e incubar em gelo durante 30 min. Adicionar 10 ml de tampão de lavagem, agitar suavemente, e centrifugar a 300 xg durante 10 min a 4 ° C. Adicionar 10 ml de tampão de lavagem, agitar suavemente, e centrifugar a 300 xg durante 10 min a 4 ° C. </li><li> Configurar umaColuna Mini-MACS, colocando uma coluna sobre o ímã MACS. Posicionar um tubo de 15 ml rotulados cónica inferior da coluna. Adicionar 1 ml de tampão de lavagem a coluna e permitir que a atravessam para o tubo de 15 ml. </li><li> Aspirar o sobrenadante e ressuspender em 1 ml de tampão de lavagem. Carregar a amostra na coluna depois de o tampão de lavagem foi executado por meio de. </li><li> Depois a amostra foi executado através da coluna, adicionar 1 ml de tampão de lavagem para lavar a coluna. Adicionar 7 ml de tampão de lavagem diretamente para a amostra coletada no tubo de 15 ml. </li><li> Contar as células usando um hemocitômetro. Transferir o volume necessário de células necessárias para a 5000000 células / cavidade em um tubo de 50 ml. Centrifugar a 300 xg durante 10 min a 4 ° C. O suplemento de meio de células B com LPS a 1: 500 e IL4 1: 1000. </li><li> Aspirar o sobrenadante e adicionar o volume apropriado de meio de células B suplementado necessário para a concentração final de um milhão de células / ml. </li><li> Células da placa em placas de 6 poços com 5 ml de células em um milhão de células / ml, e em lugar de um humidified cultura de células incubadora a 37 ° C, 5% de CO 2. </li></ol> 2. B Fixação celular <ol><li> Adicionar 50 ul de colcemide a cavidade contendo 5 ml de células B (para uma concentração final de 100 ng / ml) e incubar 1 h a 37 ° C. Pré-aquecer uma solução de KCl 75 mM num banho de água a 37 ° C. Transferir as células B a um tubo de 15 ml marcado. Cobrir a etiqueta com fita e centrifugar a 300 xg durante 10 min a 4 ° C. </li><li> Aspirar o sobrenadante deixando 1 ml no tubo e ressuspender o sedimento por pipetagem. Adicionar 5 ml de KCl a queda pré-aquecido a gota ao bater ou pulsando em um vórtice. </li><li> Adicionar 10 ml de KCl e misture invertendo. Incubar em C do banho de água 37 ° C durante 15 min. Adicione 5 gotas de fixador fresco e misture invertendo. Centrifugar a 300 xg durante 10 min a 4 ° C. Aspirar o sobrenadante deixando 1 ml no tubo e ressuspender pellet pipetando para cima e para baixo. </li><li> Adicionar 5 ml de fixador fresco gota a gota, ao bater ou pulsando em um vortex. Adicionar mais 10 ml de fixador e incubar durante 30 min à temperatura ambiente. </li><li> Centrifugar a 300 xg durante 10 min a 4 ° C. Aspirar o sobrenadante deixando 1 ml em tubos e ressuspender por pipetagem. Adicionar 14 ml de fixador fresco e centrifugar a 300 xg durante 10 min a 4 ° C. </li><li> Repita o passo 2.7 mais duas vezes. </li><li> Adicionar 14 ml de fixador fresco. Selar as tampas com Parafilm e armazenar durante a noite a -20 ° C. </li></ol> 3 Preparação de Metaphase Spreads cromossômicas <ol><li> Definir uma câmara ambiental regulada a 22,9 ° C e 52% de umidade. </li><li> Centrifugar a células B fixa a 300 xg durante 10 min a 4 ° C. Aspirar fixador deixando 70 mL no tubo e ressuspender por pipetagem. </li><li> Colocar as lâminas marcadas na câmara de humidade a um ângulo de 35 °. Queda de 35 mL de células B em ressuspensão numa lâmina e soltar duas lâminas por amostra. Permitir que os slides para secar na câmara de umidade por 30 min, em seguida, armazenar os slidesem uma caixa de lâminas a 37 ° C. </li></ol> 4. Telomere PNA FISH <ol><li> Preparação de Sonda <ol><li> Formamida desionizada pré-aquecer a 37 ° C. Misturar 2 uL de sonda ANP telómero com 7 ul de formamida desionizada pré-aquecida e incubar a 37 ° C com agitação durante 1 hora. </li><li> Pré-quente mistura principal FISH para 37 ° C. Adicionar 7 ul de pré-mistura principal aquecido FISH para a mistura a partir do passo 4.1.1 e incubar a 37 ° C durante 1-3 horas. </li><li> Desnaturar a mistura de sonda de 8 min a 80 ° C. Pré-hibridar a sonda durante 1 hora a 37 ° C. </li></ol></li><li> Pré-tratamento de Slides cromossômicas <ol><li> Pré-aquecer um frasco de deslizamento de coloração de vidro contendo 50 ml de HCl 0,01 M a 37 ° C em um banho de água. </li><li> Colocar as lâminas em um frasco de vidro para coloração de deslizamento diferentes contendo 2 x SSC durante 5 minutos à temperatura ambiente. </li><li> Adicionar 2 mL de estoque pepsina ao vidro pré-aquecido slide-coloração frasco e misturar invertendo. Transferir as lâminas para esta sl vidrojar ide-coloração e incubar 90 segundos a 37 ° C. </li><li> Lavam-se as lâminas 2x em um frasco de vidro deslizante coloração diferente contendo 1x PBS durante 5 minutos cada à temperatura ambiente, sob agitação. Em seguida, lave as lâminas por 5 min em 1x PBS / MgCl2 à temperatura ambiente, agitando. </li><li> Transferir as lâminas para um frasco de vidro para coloração deslizante contendo recém-preparado formaldeído a 1% / PBS 1x / MgCl2 durante 10 min à temperatura ambiente. </li><li> Lavar as lâminas por 5 min em um frasco de vidro deslizante coloração diferente contendo 1x PBS à temperatura ambiente, agitando. </li><li> Prepara-se uma série de etanol, tendo a desidratação de vidro separada frascos corrediça de coloração que contêm 70%, 90%, e 100% de etanol. </li><li> Transferir as lâminas para cada frasco durante 3 min, começando com 70% de etanol, em seguida etanol a 90%, e em seguida de etanol a 100%. Coloque frascos de etanol a -20 ° C quando terminar. </li><li> Permitir que as lâminas para o ar seco tocando excesso de etanol em uma toalha de papel e apoiando os slides em um ângulo de 35 °. Depois de seco, mark uma área de 18 x 18 mm para a hibridação na parte de trás das lâminas através de uma caneta de diamante. </li></ol></li><li> A desnaturação de Slides para FISH <ol><li> Aplicar formamida 120 mL de 70% deionizada / 2X SSC para uma lamela de 24 x 60 mm. Toque no slide para a lamela. Desnaturar o slide a 80 ° C sobre uma placa quente durante 90 segundos. </li><li> De forma rápida e cuidadosamente deslizar para fora da lamela e colocar a corrediça em gelo frio de etanol a 70% durante 3 min, seguido de etanol a 90%, e 100% de etanol durante 3 minutos cada. Permitir que as lâminas para o ar seco. </li><li> Prepare uma câmara úmida, alinhando uma caixa de plástico com tampa e papel de seda molhado. O papel deve ser úmido, mas não saturado com água. </li><li> Numa câmara húmida, aplicam-se a mistura de sonda de pré-recozido do passo 4.1.3 para a área marcada sobre a lâmina, coberta com uma tampa 18 x 18 mm de deslizamento e incubar a 37 ° C durante 1 hora. </li></ol></li><li> Lavagens de pós-incubação <ol><li> Pré-aquecer a 50% de formamida / 2x SSC, 1x SSC, e 4x SSC / 0,1% de Tween-20 a 45 ° em; C num banho de água. </li><li> Retire cuidadosamente as lamelas e lavar as lâminas em pré-aquecido a 50% formamida / 2x SSC 3xfor 5 min cada, no escuro e tremendo. Lavar as lâminas em pré-aquecido 1x SSC 3xfor 5 min cada, tremendo. Finalmente, lava as lâminas em pré-aquecido 4x SSC / 0,1% de Tween-20 3xfor 5 min cada. </li><li> Coloração das lâminas por 3 min em DAPI em uma luz protegida vidro slide-coloração jar. </li><li> Lavar as lâminas por 5 min em 2x SSC, tremendo. Aplicar 35 l de Mowiol meio de montagem, cubra com 24 x 60 mm lamelas e armazenar os slides a 4 ° C. </li></ol></li></ol> 5 Análise microscópica dos cromossomas <ol><li> Analisar os slides metafase utilizando um microscópio de epi-fluorescência padrão. Use uma plataforma de imagem com uma fase automatizada, como a plataforma MetaSystems Metafer para obter melhores resultados. Para cada metafase, recolher uma imagem de fluorescência para visualizar DAPI cromossomas, e uma imagem com o sinal fluorescente do telómero probe. (Por exemplo, outros Cy3- Fluors estão disponíveis e funcionam igualmente bem.) </li><li> Sobreponha as imagens com um programa de análise de imagem e analisar. </li></ol>

<ol>
	<li>Sancar, A., Lindsey-Boltz, L. A., Unsal-Kacmaz, K., Linn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+mammalian+DNA+repair+and+the+DNA+damage+checkpoints.">Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints.</a> Ann Rev Biochem. 73, 39-85 (2004).</li><li>Sperka, T., Wang, J., Rudolph, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+checkpoints+in+stem+cells,+ageing+and+cancer.">DNA damage checkpoints in stem cells, ageing and cancer.</a> Nature reviews Mol Cell Biol. 13 (9), 579-590 (2012).</li><li>Alexandrov, L. B., 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=Signatures+of+mutational+processes+in+human+cancer.">Signatures of mutational processes in human cancer.</a> Nature. 500 (7463), 415-421 (2013).</li><li>Bunting, S. F., 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=53BP1+inhibits+homologous+recombination+in+Brca1-deficient+cells+by+blocking+resection+of+DNA+breaks.">53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks.</a> Cell. 141 (2), 243-254 (2010).</li><li>Padilla-Nash, H. M., Barenboim-Stapleton, L., Difilippantonio, M. J., Ried, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+karyotyping+analysis+of+human+and+mouse+chromosomes.">Spectral karyotyping analysis of human and mouse chromosomes.</a> Nat Protoc. 1 (6), 3129-3142 (2006).</li><li>Callen, E., 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=Chimeric+IgH-TCRalpha/delta+translocations+in+T+lymphocytes+mediated+by+RAG.">Chimeric IgH-TCRalpha/delta translocations in T lymphocytes mediated by RAG.</a> Cell Cycle. 8 (15), 2408-2412 (2009).</li><li>Boboila, C., 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=Alternative+end-joining+catalyzes+robust+IgH+locus+deletions+and+translocations+in+the+combined+absence+of+ligase+4+and+Ku70.">Alternative end-joining catalyzes robust IgH locus deletions and translocations in the combined absence of ligase 4 and Ku70.</a> Natl Acad Sci USA. 107 (7), 3034-3039 (2010).</li><li>Benn, P., Delach, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+lymphocyte+culture+and+chromosome+analysis.">Human lymphocyte culture and chromosome analysis.</a> Cold Spring Harb Protoc. 2008, (2008).</li><li>Nguyen, H. N., Reijo Pera, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metaphase+spreads+and+spectral+karyotyping+of+human+embryonic+stem+cells.">Metaphase spreads and spectral karyotyping of human embryonic stem cells.</a> Cold Spring Harb Protoc. 2008, (2008).</li><li>Schreck, R. R., Disteche, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+4.">Chapter 4.</a> Chromosome banding techniques. Unit 2, (2001).</li><li>Gilley, D., 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=DNA-PKcs+is+critical+for+telomere+capping.">DNA-PKcs is critical for telomere capping.</a> Proc Natl Acad Sci USA. 98 (26), 15084-15088 (2001).</li><li>Bolzan, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+aberrations+involving+telomeres+and+interstitial+telomeric+sequences.">Chromosomal aberrations involving telomeres and interstitial telomeric sequences.</a> Mutagenesis. 27 (1), 1-15 (2012).</li><li>Bolzan, A. D., Telomeres Bianchi, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=interstitial+telomeric+repeat+sequences+and+chromosomal+aberrations.">interstitial telomeric repeat sequences and chromosomal aberrations.</a> Mutat Res. 612 (3), 189-214 (2006).</li><li>Poon, S. S., Lansdorp, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+18.">Chapter 18.</a> Quantitative fluorescence in situ hybridization (Q-FISH). Unit 18 14, (2001).</li><li>Morales, J. C., 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=53BP1+and+p53+synergize+to+suppress+genomic+instability+and+lymphomagenesis.">53BP1 and p53 synergize to suppress genomic instability and lymphomagenesis.</a> Proc Natl Acad Sci USA. 103 (9), 3310-3315 (2006).</li><li>Halaby, M. J., 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=Synergistic+interaction+of+Rnf8+and+p53+in+the+protection+against+genomic+instability+and+tumorigenesis.">Synergistic interaction of Rnf8 and p53 in the protection against genomic instability and tumorigenesis.</a> PLoS Genet. 9 (1), e1003259 (2013).</li></ol>

Os autores não têm interesses conflitantes.

Considerando que os linfócitos B activados são particularmente adequados para a preparação de barrar cromossómicas mitóticas, outros tipos de células podem também ser utilizados. Linfócitos T partilhar muitas das vantagens de células B, uma vez que podem ser purificadas a partir dos gânglios linfáticos ou do baço, e têm um elevado índice mitótico, quando estimulada por crescimento em meio contendo mitogénios adequadas 8. As células-tronco embrionárias (células ES) também são adequados pa...

A correction was made to: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH.&#160;Table 1 (the list of solutions to prepare) was omitted from the Protocol section. The contents of Table 1 are as follows:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Reagents</td>
			<td>Comments</td>
			<td>Storage</td>
		</tr>
		<tr>
			<td rowspan="3">B Cell Wash Buffer</td>
			<td>500 ml 1x Hanks Balanced Salt Solution</td>
			<td rowspan="3">Filter through 0.22 &#956;m stericup filtration unit</td>
			<td rowspan="3">4 &#176;C</td>
		</tr>
		<tr>
			<td>5 ml 50 &#176;C heat inactivated FCS</td>
		</tr>
		<tr>
			<td>5 ml 100x Pen/Strep</td>
		</tr>
		<tr>
			<td rowspan="8">B Cell Medium</td>
			<td>418.5 ml RPMI-1640</td>
			<td rowspan="8">Filter through 0.22 &#956;m stericup filtration unit</td>
			<td rowspan="8">4 &#176;C</td>
		</tr>
		<tr>
			<td>50 ml 50 &#176;C heat inactivated FCS</td>
		</tr>
		<tr>
			<td>5 ml 100x Pen/Strep</td>
		</tr>
		<tr>
			<td>5 ml glutamine</td>
		</tr>
		<tr>
			<td>5 ml nonessential amino acids</td>
		</tr>
		<tr>
			<td>5 ml sodium pyruvate</td>
		</tr>
		<tr>
			<td>1.8 ml 14.2 M 2-mercaptoethanol</td>
		</tr>
		<tr>
			<td>5 ml 1M HEPES</td>
		</tr>
		<tr>
			<td>Fixative</td>
			<td>3:1 v/v methanol/glacial acetic acid</td>
			<td></td>
			<td>Make fresh</td>
		</tr>
		<tr>
			<td rowspan="2">0.075 M KCl</td>
			<td>1.395 g KCl</td>
			<td rowspan="2"></td>
			<td rowspan="2">Room Temperature</td>
		</tr>
		<tr>
			<td>250 ml distilled H2O</td>
		</tr>
		<tr>
			<td rowspan="3">FISH Master Mix</td>
			<td>40 ml 50% Dextran sulfate</td>
			<td rowspan="3">Vortex the solution and leave in a shaking platform overnight</td>
			<td rowspan="3">Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>20 ml 20x SSC</td>
		</tr>
		<tr>
			<td>40 ml sterile distilled H2O</td>
		</tr>
		<tr>
			<td rowspan="3">70% Formamide/2x SSC</td>
			<td>10 ml 20x SSC</td>
			<td rowspan="3">Adjust to pH 7 with 1M HCl</td>
			<td rowspan="3">Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>20 ml distilled H2O</td>
		</tr>
		<tr>
			<td>70 ml deionized formamide</td>
		</tr>
		<tr>
			<td rowspan="2">0.01 M HCl</td>
			<td>1 ml 1M HCl</td>
			<td rowspan="2">Adjust to pH 2.0</td>
			<td rowspan="2">Room Temperature</td>
		</tr>
		<tr>
			<td>99 ml distilled H2O</td>
		</tr>
		<tr>
			<td rowspan="2">1x PBS/MgCl2</td>
			<td>50 ml 1M MgCl2</td>
			<td rowspan="2"></td>
			<td rowspan="2">Room Temperature</td>
		</tr>
		<tr>
			<td>950 ml 1x PBS</td>
		</tr>
		<tr>
			<td rowspan="3">50% Formamide/2x SSC</td>
			<td>20 ml 20x SSC</td>
			<td rowspan="3">Adjust to pH 7.25 
			Formamide used in this step does not have to be deionized.</td>
			<td rowspan="3">Make fresh</td>
		</tr>
		<tr>
			<td>80 ml distilled H2O</td>
		</tr>
		<tr>
			<td>100 ml formamide</td>
		</tr>
		<tr>
			<td rowspan="3">4x SSC/0.1% Tween20</td>
			<td>50 ml 20x SSC</td>
			<td rowspan="3"></td>
			<td rowspan="3">Room Temperature</td>
		</tr>
		<tr>
			<td>200 ml distilled H2O</td>
		</tr>
		<tr>
			<td>250 &#956;l Tween20</td>
		</tr>
		<tr>
			<td rowspan="2">DAPI staining solution (80 ng/ml)</td>
			<td>40 &#956;l 0.2mg/ml DAPI stock</td>
			<td rowspan="2"></td>
			<td rowspan="2">4 &#176;C in light protected staining jar</td>
		</tr>
		<tr>
			<td>100 ml 2x SSC</td>
		</tr>
		<tr>
			<td rowspan="5">Mowiol Mounting Medium</td>
			<td>6 g glycerol</td>
			<td rowspan="5">Incubate at 50 &#176;C with stirring for 2hrs</td>
			<td rowspan="5">Aliquot and store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>2.4 g Mowiol 4-88</td>
		</tr>
		<tr>
			<td>6 ml distilled H2O</td>
		</tr>
		<tr>
			<td>12 ml 0.2 M TrisCl pH 8.5</td>
		</tr>
		<tr>
			<td>230 &#956;l 1% Thimerosal (w/v in water)</td>
		</tr>
	</tbody>
</table>
Table 1. List of solutions.&#160;

A correction was made to: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH.

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

O cancro é causado pela acumulação de mutações que afectam os genes que regulam o crescimento de células normais. A mutação é uma conseqüência de alterações na estrutura e seqüência do genoma causada por danos ao DNA. Danos do ADN pode ocorrer através de uma variedade de processos, incluindo agentes exógenos, tais como radiação ionizante e, como um subproduto do metabolismo celular normal, tal como a desaminação espontânea de bases de nucleótidos que ocorram danos ou por contacto com espécies de oxigénio reactivas 1. Embora as células de mamíferos possuem uma gama de actividades de reparação, que pode reverter danos ao DNA ou restaurar a seqüência em locais de quebra, no entanto, mutações se acumulam ao longo do tempo de vida de uma célula. Danos de DNA pode, além disso, contribuir para a senescência e a perda de potência de células-tronco, dois processos que estão associados com a doença associada ao envelhecimento 2. Compreender a reparação de danos ao DNA é, portanto, de importância central na abordagem de dois sinalquestões ificant em saúde pública. Uma evidência crescente sugere que as vias de reparação do ADN de mamíferos pode contribuir para a evolução do genoma de células de cancro de 3,4, o que torna ainda mais fundamental para compreender os processos envolvidos na supressão da mutação a nível molecular. A visualização direta de aberrações cromossômicas é um meio poderoso e quantitativos de determinar a extensão da instabilidade genômica em um tipo particular de célula. Cromossomos condensados ​​de células em metáfase pode ser isolado e inspecionados utilizando luz ou microscopia fluorescente. Tais abordagens citogenéticas têm sido, na prática há várias décadas e pode ser utilizado para demonstrar a aparência de translocação ou de determinados tipos de aberrações cromossómicas relacionadas com a perda de actividade de reparação de ADN. O protocolo presta-se a várias extensões possíveis: os cromossomas podem ser marcadas com sondas para cariotipagem espectral (CÉU) ou multicolor hibridização in situ fluorescente(MFISH) para identificar translocações 5,6. Essas técnicas também permitem a frequência de translocações cromossômicas ea estrutura de translocações cromossômicas complexas para ser determinada, que fornece informações adicionais para além do que é possível com este protocolo. Alternativamente, as sondas específicas para a sequência pode ser gerada e utilizada para testar a frequência de quebra de ADN em locais genómicos seleccionados 7. Neste protocolo, descrevemos a preparação do cromossomo metaphase espalha a partir de linfócitos B. Um ácido nucleico (PNA) péptido sonda fluorescente marcada por repetições teloméricas é utilizado, de forma eficiente, que marca telómeros em cromossomas da metafase espalha Este protocolo tem várias vantagens. As células B podem ser induzidas a crescer em alto índice mitótico de modo que os diferenciais de alta qualidade pode ser produzido de forma consistente. Células B de camundongos geneticamente modificados também são muito menos propensos a conter mutações genéticas secundárias que podem confundir a análise da contribuiçãode genes específicos para a integridade genómica. A abordagem PNA-FISH pode ser concluído em um dia, e permite marcar mais precisa de quebras cromossômicas. Usando essa abordagem, particularmente em combinação com equipamento específico descrito no presente protocolo, é possível produzir muito consistentes, spreads de alta qualidade e analisar rapidamente a taxa eo tipo de instabilidade genômica.

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rapid analysis of chromosome aberrations in mouse b lymphocytes by pna-fish

Reparo do DNA defeituoso leva ao aumento da instabilidade genômica, que é a causa de mutações que levam à tumorigênese. A análise da frequência e tipo de aberrações cromossômicas em diferentes tipos de células permite defeitos nas vias de reparo de DNA a ser elucidado. Biologia de reparo do DNA em mamíferos Entendimento foi muito ajudado pela produção de camundongos com nocautes em genes específicos. O objetivo deste protocolo é quantificar instabilidade genômica em linfócitos B de ratinho. Rotulagem dos telômeros, utilizando sondas PNA-FISH (ácido nucleico peptídico - hibridização in situ fluorescente) facilita a análise rápida de instabilidade genômica dos spreads metáfase de cromossomos. As células B têm vantagens específicas em relação a fibroblastos, porque eles têm ploidia normal e um índice mitótico elevado. Cultura de curto prazo das células B, portanto, permite a medição precisa da instabilidade genômica em uma população celular primária que é susceptível de ter menos mutação genética secundários do que o que é normalmente encontrado em fibroblastos transformados ou linhas celulares de doentes.

Cromossomos metafásicos derivadas de uma célula devem formar um conjunto discreto contendo 40 cromossomos (se estiver usando células de camundongo) (Figura 1A). Cada cromossoma tem um centrómero em uma extremidade, que é visível como uma esfera de luz azul. Em cromossomos normais, dois sinais dos telômeros são vistos no fim das cromátides e dois sinais de cada centrômero. Os núcleos interfásicos vai estar presente na lâmina bem. Estes serão visíveis como esferas azuis, com os sinais dos t...

Watch this Scientific Journal Video about Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH at JoVE.com

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

Vias de reparo de DNA são essenciais para a manutenção da integridade genômica e mutação prevenção e câncer. O objetivo deste protocolo é quantificar instabilidade genômica por observação direta de aberrações cromossômicas em metáfase se espalha a partir de células de rato B usando hibridização in situ fluorescente (FISH) para repetições de DNA telomérico.

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 ...

rapid-analysis-chromosome-aberrations-mouse-b-lymphocytes-pna

Research

JoVE Journal

Immunology and Infection

16.8K visualizações.  Rutgers, the State University of New Jersey.  Vias de reparo de DNA são essenciais para a manutenção da integridade genômica e mutação prevenção e câncer. O objetivo deste protocolo é quantificar instabilidade genômica por observação direta de aberrações cromossômicas em metáfase se espalha a partir de células de rato B usando hibridização in situ fluorescente (FISH) para repetições de DNA telomérico. 

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

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

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Métodos para avaliar Mediated Beta Morte celular por linfócitos T citotóxicos

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

O isolamento de linfócitos de rato Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many proteins. Classic imaging techniques have been used to study these phenomena. However, they required the manual acquisition of many cells followed by time consuming quantification of the morphology and the co-localization of the staining of tens of cells. Here, a rapid and powerful method is described to study these phenomena on samples consisting of several thousands of cells using an imaging flow cytometry technology that combines the advantages of a microscope with those of a cytometer. Using T lymphocytes stimulated with CCL19 and staining for MHC Class I molecules and filamentous actin, a gating strategy is presented to measure simultaneously the degree of shape alterations and the extent of co-localization of markers that are affected by CCL19 signaling. Moreover, this gating strategy allowed us to observe the segregation of filamentous actin (at the front) and phosphorylated Ezrin-Radixin-Moesin (phospho-ERM) proteins (at the rear) in polarized T cells after CXCL12 stimulation. This technique was also useful to observe the blocking effect on polarization of two different elements: inhibition of actin polymerization by a pharmacological inhibitor and expression of mutants of the Par6/atypical PKC signaling pathway. Thus, evidence is shown that this technique is useful to analyze both morphological alterations and protein redistributions.

Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

Análise rápida e robusta de Celular e Molecular Polarização Induzida por Chemokine Signaling

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

Detecção de True IgE-expressando Rato B células da linhagem

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

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

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

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 ...

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 ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Rápida Detecção Molecular e diferenciação de Influenza vírus A e B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

Difusão molecular em membranas plasmáticas de linfócitos primários medida por fluorescência Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

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., ...