Agradecemos a Steve McBryant e Jeff Hansen para ajudar com ultracentrifugação e Kiser Patti para ajudar no tratamento de mouse. O Instituto Nacional de Doenças Neurológicas e Derrames no National Institutes of Health, conceder 5R01NS056379-02 financiado este trabalho.

<html> 1. Purificação e Rotulagem Rods Prion Este protocolo foi adaptado de um 18 previamente publicados <ol><li> Homogeneizar 100 gramas de prion infectados tecido cerebral em 900 mL de tampão de homogeneização gelada (HB, 1X PBS contendo sacarose 320 mM, 150 mM NaCl e 4 mM EDTA) 1 min na velocidade máxima em um liquidificador comercial, em seguida, 2 min no gelo. Repita três vezes. </li><li> Centrífuga homogeneizado por 10 min a 3000 xg e 4 ° C. Retire e guarde o sobrenadante no gelo. Re-suspender sedimento em 1L HB e repita os passos 1.1 e 1.2. </li><li> Sobrenadantes piscina e centrifugar por 60 min a 100.000 xg, 0 ° C. Elimine o sobrenadante. </li><li> Re-suspender pellet em 100 ml de 1X Tris salina tamponada (10 mM Tris-HCl, 0,1 mM NaCl e 1 mM EDTA) contendo 2% Triton X-100 (TBST). Concentração de proteína pela estimativa Bradford ou ensaios semelhantes e ajustar a 5 mg / mL em TBST. Frio no gelo por 30 min. </li><li> Centrifugar por 30 min a 100.000 xg, 0 ° C. Elimine o sobrenadante e lavar pellet com 50 TBST mL, em seguida, 100 mL TBS. Re-suspender pellet em 100 ml de 1X PBS contendo 1% sarcosyl e coquetel inibidor de protease e mexa por 120 min a 37 ° C. </li><li> Camada de amostra em 900 mL de 320 mM de sacarose e centrifugar por 60 min a 100.000 xg, 10 ° C. Elimine o sobrenadante e re-suspensão sedimento em 10 mL 2,3 M NaCl, sarcosyl 5%. </li><li> Sonicate amostra de 5 x 40 segundos na potência máxima de 70% em intervalos de 1 min no gelo. </li><li> ML de amostra de uma alíquota em tubos eppendorf e centrifugar por 15 minutos a 13.000 xg, 4oC. Lave duas vezes com TBS pellets e armazenar @ -70 ° C ou conjugado fluorocromo no passo 1.9. </li><li> Conjugado de um tubo de varas purificada prion usando 1 frasco de 649 DyLight fluorocromo de acordo com o protocolo do fabricante. </li><li> Troca de buffer rotulagem DyLight para PBS por diálise ou centrifugação através de colunas de filtro. Loja em 20 mL alíquotas a -70 ° C ao abrigo da luz até que esteja pronto para uso. </li></ol> 2. Inoculação príon celular e Recuperação Esta seção abrange a inoculação intraperitoneal prion e recuperação de células do peritônio, linfonodos mediastinais e mesentéricos e baço. <ol><li> Diluir varas prion fluorescentes 1:50 em PBS imediatamente antes da utilização. Rato Scruff pela pele do pescoço dorsal com o polegar eo dedo indicador, delicadamente por sua vez do mouse para enfrentá-lo e restringir cauda com pinky dedo. Supinate mouse, elevando o tronco traseiras acima da cabeça e segurando-o levemente de cabeça para baixo. </li><li> Usando uma seringa de insulina 30G, injectar 100 mL de varas prion fluorescente 1 cm lateral da linha média e 1-2 cm anterior à articulação sacroilíaca. Insira a agulha 1 centímetro através da pele direcionado 45 ° medialmente. Use 1:50 diluição de partículas fluorescentes ou sozinho PBS como controle. Incubar camundongos por tempo determinado antes da análise. </li><li> Euthanize ratos de acordo com o cuidado de animais aprovados e usam protocolos da comissão. Injectar 10 ml de PBS para a cavidade peritoneal, utilizando uma seringa e agulha 12 mL 25G. Agite levemente carcaça longitudinalmente para um minuto. Recuperar suspensão de células de peritônio usando a mesma seringa e uma agulha de 16G em um tubo cônico de 15 mL e reserva no gelo, enquanto a dissecação de carcaça continua. </li><li> Molhada no lado ventral da carcaça cuidadosamente com etanol 70%. Levante a pele na linha média logo abaixo da caixa torácica com uma pinça e corte uma pequena incisão através da pele com uma tesoura. A partir daí, fazer uma incisão centímetros 05/03 longa através da pele em ambos os sentidos a cabeça ea cauda, ​​em seguida, dois 2 cm de incisões lateralmente da linha média de cada extremidade. Retire a pele e pinos para revelar as cavidades peritoneal e torácica. Corte cuidadosamente através das membranas translúcidas finas ao redor do peritônio e refletem o tórax para revelar o mediastino. </li><li> Encontrar e remover 2-4 linfonodos mediastinais, ligeiramente dorsal e lateralmente adjacentes para o timo, medialmente adjacente à artéria braquiocefálica, perto do lado ventral da traquéia. Encontrar e remover até seis gânglios linfáticos mesentéricos, embutidos no tecido macio branco mesentérica, que ancora o intestino à parede abdominal posterior. Encontrar e remover o baço, um longo, vermelho escuro, órgão localizado apenas a lateral da linha média dorsal e para o intestino, no lado esquerdo do abdômen. Linfonodo de reserva em 1 mL RPMI 1640, em gelo até dissecção é completa. </li><li> Macerado e pressione gânglios linfáticos através de uma peneira de malha 40 mM em 3 mL de RPMI em uma placa de Petri de plástico usando o êmbolo de uma seringa de 1 mL. Enxágüe com um filtro de 2 mL de RPMI e transferir a suspensão celular 5 mL para um tubo cônico de 15 mL. Lavar a placa de Petri com um adicional de 5 mL de RPMI e transferência para o mesmo tubo. Centrifugar 5 minutos a 250 xg, elimine o sobrenadante, ressuspender o pellet celular em 1 mL FACS tampão (1X PBS, 1% de albumina bovina sérica e 10 mM EDTA) e transferir para um tubo Eppendorf 1,5 mL. </li></ol><html> ove_title &quot;Coloração&gt; 3. Celular e Citometria de Fluxo O seguinte é um protocolo de coloração típica para a identificação de subconjuntos de células imunes utilizando anticorpos fluorescentes contra bem caracterizados marcadores de superfície das células imunológicas. Todos os anticorpos foram diluídos em tampão FACS imediatamente antes da utilização. <ol><li> Contagem de células e alíquota de 10 6 células em tubos Eppendorf. Células de centrifugação (ver 2.7) e lavar pellet 2X com um tampão FACS mL. </li><li> Incubar as células 20 min no gelo em 100 mL de 2 ng / mL do mouse anti-rato CD16/32 para bloquear os receptores Fc endógena. Pellet e células de lavagem com tampão FACS. </li><li> Adicionar 100 mL de 1ng / mL apropriado anticorpos fluorescentes (y) s para cada amostra e incubar no gelo por uma hora. Adicionar 100 mL de tampão FACS sozinho para as células controle. Pellet e células lavar duas vezes com tampão FACS. </li><li> Re-suspender as células em 1 mL de tampão ACK (150 mM NH 4 Cl, 1 mM KHCO 3 e 0,1 mM EDTA) Incubar em gelo por 1 min para lisar as células vermelhas do sangue. Pellet e células de lavagem com tampão FACS. Re-suspender as células em um buffer de FACS mL. </li><li> Coletar dados a partir de células usando um citômetro de fluxo capazes de detecção e análise multicolor, tipicamente com 405, 488 e 633 nm de excitação e lasers 09:55 detectores de emissão de fluorescência. </li></ol> 4. Resultados representativos: Nós purified varas prion de crude, prion infectados homogenato de cérebro (Figura 1) solubilização Detergente e ultracentrifugação dos alces E2 cérebro homogenato (faixas 1 e 2) grandemente enriquecido para hastes de prion (compare raias 1 e 3), que também foi protease K resistente (faixa 4). Curiosamente, não digerido varas prion purificada exibiu um perfil muito semelhante à glycoform homogeneizado digerida E2 cérebro, indicando enriquecimento dramático para príons. Homogenato de cérebro de forma idêntica tratados normais não produziu PK-resistente PrP C (faixas 5 e 6). Análise de citometria de fluxo de populações de células específicas demonstram que as células apresentadoras de antígenos para casa para o peritônio em duas horas e reter partículas fluorescentes e barras de príon, como evidenciado por fluorescência aumentou dramaticamente ao longo controles PBS injetado (Figura 2). Não células colhidas do peritônio de PBS-tratados controles exibidos fluorescência acima de fundo (Figs. 2A-G), enquanto as células de talão (Figs. 2H-N) e prion (Figs. 2O-U)-inoculados camundongos exibido fluorescência significativa, especialmente em monócitos (Figs. 2I e P), células dendríticas (Figs. 2K e R) e macrófagos (Figs. 2L e S), e alguns neutrófilos (Figura 2J e Q) e menos células B (Figura 2T). Grânulo e Prion-rolamento monócitos, células dendríticas e macrófagos também foram encontrados nos gânglios linfáticos do mediastino duas horas após a inoculação (Figs. 2AD e AK, AF e AG e AM e AN, respectivamente), enquanto poucas ou nenhuma PrP CWD-bearing neutrófilos , as células B ou T foram encontradas lá (Figs. 2AE e AL, AH e AG e AI e AP, respectivamente). Nós não detectou contas ou varas príon no baço ou linfonodos mesentéricos (dados não mostrados). In totum, estes dados demonstram que o tráfego de células do sistema imunológico prions de forma muito semelhante aos antígenos de outras partículas. <table border="1"><tbody><tr><td> REAGENT </td><td> Fluorocromo </td><td> LASER excitação (nm) </td><td> Pico de emissão (nm) </td></tr><tr><td> Hastes príon </td><td> DyLight 649 </td><td> 633 </td><td> 674 </td></tr><tr><td> 1 mM contas </td><td> AlexaFluor 660 </td><td> 633 </td><td> 685 </td></tr><tr><td> Anticorpos </td><td></td><td></td><td></td></tr><tr><td> CD11b </td><td> eFluor 450 </td><td> 405 </td><td> 450 </td></tr><tr><td></td><td> Ficoeritrina-Cy7 </td><td> 488 </td><td> 760 </td></tr><tr><td> CD11c </td><td> R-Ficoeritrina </td><td> 488 </td><td> 575 </td></tr><tr><td></td><td> Ficoeritrina-Cy7 </td><td> 488 </td><td> 760 </td></tr><tr><td> Ly6C </td><td> Fluoroisothiocyante </td><td> 488 </td><td> 518 </td></tr><tr><td> Ly6G </td><td> R-Ficoeritrina </td><td> 488 </td><td> 575 </td></tr><tr><td> CD21 </td><td> DyLight 488 </td><td> 488 </td><td> 518 </td></tr><tr><td> B220 </td><td> Allophycocyanin-Cy7 </td><td> 633 </td><td> 785 </td></tr><tr><td> CD3 </td><td> R-Ficoeritrina </td><td> 488 </td><td> 575 </td></tr></tbody></table>&quot;&gt; Tabela 1. Propriedades espectrais de reagentes fluorescentes <img src="/files/ftp_upload/2349/2349fig1.jpg" alt="Figura 1" /> Figura 1. Purificação de varas prion de homogenato de cérebro infectado. Usamos um homogenato de cérebro de 10% bruto de um cervo CWD-infectados (E2, faixas 1 e 2) como matéria-prima da qual purificada varas prion agregado (faixas 3 e 4). Bruta e purificada materiais apresentaram resistência característica a protease K tratamento (faixas 1-4), enquanto homogenato de cérebro normal não (faixas 5 e 6). Marcadores de peso molecular são mostrados na Kd à esquerda da mancha. BH, homogenato de cérebro. <img src="/files/ftp_upload/2349/2349fig2.jpg" alt="Figura 2" /> Figura 2. Citometria de fluxo imunológico prions tráfico células do peritônio para os gânglios linfáticos do mediastino duas horas após a inoculação. PBS (painéis AG e V-AB), partículas fluorescentes (HN e AC-AI) ou varas de Prion (OU e AJ-AP) foram injetado no peritônio de camundongos e células colhidas de lavado peritoneal (AU) ou linfonodos mediastinais (V-AP), duas horas mais tarde. Gráficos nas células primeira coluna show de camundongos tratados com PBS (painéis A e V), partículas fluorescentes (painel H e AC) e hastes de prion (painéis S e AJ). Células fluorescentes (pontos vermelhos ou verdes) e total de células (pontos cinza) são plotados para mostrar tamanho relativo de dispersão (para frente), granularidade de dispersão (lateral) e proporção do total de células vivas que apresentam fluorescência. Um número significativo de células característica de granulócitos retidos partículas fluorescentes e barras. Células também foram coradas com anticorpos contra marcadores de células imunes de superfície e fechado para LYG-Ly6C + monócitos (painéis B, I, P, W, AD e AK), Ly6c-CD11c-CD11b + Ly6G + neutrófilos (C, J, Q, X, AE e AL), Ly6G-Ly6C-CD11b + CD11c + células dendríticas (D, K, R, Y, AF e AM), Ly6G-Ly6C-CD11c-CD11b + macrófagos, (E, L, S, Z, AG e AN), CD3 -B220 + CD21 + células B (F, M, T, AA, AH e AO) e CD21-B220-CD3 + células T (G, N, U, AB, AI e AP). Monócitos, células dendríticas e macrófagos retidas partículas fluorescentes e barras de prion na cavidade peritoneal (painéis I e P, K e L e R e S, respectivamente) e transportou-os para os gânglios linfáticos do mediastino (painéis AD e AK, AF e AG e AM e AN, respectivamente), demonstrando absorção semelhantes e tráfico destas duas partículas. Neutrófilos retidos uma quantidade significativa de contas (J) e hastes menos (Q), mas não conseguiu entregá-los para os nódulos linfáticos (AE e AL). Células B retido e transportado algumas hastes (T e AO), mas praticamente nenhum contas (M e AH). Células T traficadas nem contas nem varas (N, U, AI e AP).

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+and+oral+transmission+of+prion+diseases.">Dendritic cells and oral transmission of prion diseases.</a> Adv Drug Deliv Rev. 56, 901-901 (2004).</li><li>Ano, Y., Sakudo, A., Nakayama, H., Onodera, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+and+dynamics+of+infectious+prion+protein+in+the+intestine.">Uptake and dynamics of infectious prion protein in the intestine.</a> Protein Pept Lett. 16, 247-247 (2009).</li><li>Aucouturier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infected+splenic+dendritic+cells+are+sufficient+for+prion+transmission+to+the+CNS+in+mouse+scrapie.">Infected splenic dendritic cells are sufficient for prion transmission to the CNS in mouse scrapie.</a> J Clin Invest. 108, 703-703 (2001).</li><li>Huang, F. 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Aqui demonstramos um protocolo para a marcação e rastreamento de prions in vivo que muito facilita o monitoramento de eventos no início de príons periféricos. Este protocolo aumenta muito em tentativas passadas de captação de prion de monitoramento in vitro 9 e in vivo 3,12 por pré-inóculo rotulagem prion altamente enriquecido para diferenciá-lo de forma inequívoca endógena PrP C. Porque príons infecciosos e PrP C compartilhar a mesma se...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>CWD-infected elk brain</td>
<td>&nbsp;</td>
<td>Private elk farm in Colorado</td>
<td>&nbsp;</td>
<td>Use any non-human prion-infected brain</td>
</tr>
<tr>
<td>Blender</td>
<td>&nbsp;</td>
<td>Oster</td>
<td>6694-015</td>
<td>Use any commercial blender</td>
</tr>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>Sorvall</td>
<td>SS34 rotor</td>
<td>Use any centrifuge /rotor that can reach 3000 x g and hold &ge; 500 ml volumes</td>
</tr>
<tr>
<td>Ultracentrifuge</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>50.2 Ti rotor</td>
<td>Use any ultracentrifuge /rotor that can reach 100,000 x g and hold &ge; 500 ml volumes</td>
</tr>
<tr>
<td>Bradford Reagent</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>B6916</td>
<td>&nbsp;</td>
<tr>
<td>Complete mini protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11 836 170 001</td>
<td>&nbsp;</td>
<tr>
<td>Sonicator</td>
<td>&nbsp;</td>
<td>Misonix</td>
<td>MP4000X</td>
<td>Use any horn or probe sonicator set to ~70% max power</td>
</tr>
<tr>
<td>DyLight antibody Labeling kit</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>53050</td>
<td>&nbsp;</td>
<tr>
<td>microcentrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>55430R</td>
<td>Use any refrigerated microcentrifuge that can achieve 13,000x g</td>
</tr>
<tr>
<td>centrifugal filter columns</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>Microcon YM-100</td>
<td>Use any filter or dialysis membrane with 100 Kd molecular weight cutoff</td>
</tr>
<tr>
<td>8-40 week-old FVB mice</td>
<td>&nbsp;</td>
<td>Charles River</td>
<td>207</td>
<td>Use any inbred mouse strain</td>
</tr>
<tr>
<td>1 &mu;m red fluorescent beads</td>
<td>&nbsp;</td>
<td>Phosphorex</td>
<td>2307</td>
<td>Use any fluorescent bead &le; 10 &mu;m</td>
</tr>
<tr>
<td>RPMI 1640 medium </td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11875-093</td>
<td>&nbsp;</td>
<tr>
<td>40 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>fluorescent antibodies</td>
<td>&nbsp;</td>
<td>BD pharmingen</td>
<td>Various</td>
<td>Use any fluorescent antibody appropriate for your application.</td>
</tr>
<tr>
<td>flow cytometer</td>
<td>&nbsp;</td>
<td>Dakocytomation</td>
<td>CyanADP</td>
<td>Use any flow cytometer capable of multicolor fluorescence detection</td>
</tr>		</tbody>
		</table>
		</div>

monitoring immune cells trafficking fluorescent prion rods hours after intraperitoneal infection

Presença de uma forma anormal de uma proteína prion host-codificado (PrPC) que é resistente à protease, patológicas e infecciosas caracteriza doenças de príon, como a doença crônica Wasting (CWD) de cervídeos e scrapie em ovinos. A hipótese de Prion afirma que esta conformação anormal constitui a maior parte ou todo o príon infeccioso. O papel do sistema imunológico em eventos precoces na patogênese da prion periféricos tem sido convincentemente demonstrada por CWD e 1-3 scrapie. Transgênicos e farmacológicas estudos em ratos revelou um importante papel do sistema complemento na manutenção e replicando prions início após a infecção 4-6. In vitro e in vivo estudos também observaram retenção de prion pelas células dendríticas 10/07, apesar de seu papel no tráfico permanece claro 11-16. Macrófagos têm igualmente sido implicados na patogênese da prion cedo, mas esses estudos concentraram-se em eventos que ocorrem semanas após a infecção 3,11,17. Estes estudos anteriores também sofrem com o problema da diferenciação entre endógena PrP C e príons infecciosos. Aqui nós descrevemos uma abordagem imparcial semiquantitativo para avaliar a absorção de príon eo tráfico a partir do sítio de inoculação de células do sistema imunológico recrutados lá. Hastes prion agregados foram purificados a partir de homogenato de cérebro infectado por solubilização de detergente não agregados de proteínas e ultracentrifugação através de uma almofada de sacarose. Eletroforese em gel de poliacrilamida, coomassie coloração azul e recuperação de mata-borrão ocidental confirmados de varas prion altamente enriquecido na fração peletizada. Hastes príon foram fluorocromo marcado por via intraperitoneal, em seguida, injetadas em ratos. Duas horas mais tarde células imunológicas de lavado peritoneal, baço e gânglios linfáticos do mediastino e mesentéricos foram ensaiadas para a retenção de prion haste e subpopulações de células identificadas por multicolor citometria de fluxo utilizando marcadores para monócitos, neutrófilos, células dendríticas, macrófagos e células B e T. Este ensaio permite o monitoramento direto da primeira vez que células do sistema imunológico aquisição e prions tráfico in vivo dentro de horas após a infecção. Este ensaio também diferencia claramente infecciosas, prions agregadas de PrPC normalmente expressa em células do hospedeiro, que pode ser difícil e levar a problemas de interpretação de dados em sistemas de ensaio de outros. Este protocolo pode ser adaptado para outras rotas de inoculação (oral, intravenosa, subcutânea e intranervous, por exemplo) e antígenos (grânulos conjugados, patógenos bacterianos, virais e parasitárias e proteínas, o ovo) também.

Watch this Scientific Journal Video about Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection at JoVE.com

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Aqui nós descrevemos um ensaio de romance para o monitoramento e captação de prion tráfico por células do sistema imunológico imediatamente após a inoculação intraperitoneal de purificação e fluorescente rotulagem varas prion agregados de material cerebral infectado acompanhamento da sua absorção e movimento a partir do local da injeção e caracterizar as células mediadoras desses eventos.

Monitoramento células imunes Tráfico Rods Horas Prion fluorescente após a infecção intraperitoneal

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

monitoring-immune-cells-trafficking-fluorescent-prion-rods-hours

Research

JoVE Journal

Immunology and Infection

11.4K visualizações.  Colorado State University. Aqui nós descrevemos um ensaio de romance para o monitoramento e captação de prion tráfico por células do sistema imunológico imediatamente após a inoculação intraperitoneal de purificação e fluorescente rotulagem varas prion agregados de material cerebral infectado acompanhamento da sua absorção e movimento a partir do local da injeção e caracterizar as células mediadoras desses eventos.

Vídeo: Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Medir a atividade das calpaínas em células fixas e Vivendo por citometria de fluxo

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Imunologia e Infecção

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Sete Passos para Stellate Células

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Análise por Citometria de fluxo das células imunológicas Dentro aortas murino

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Isolamento de células do sistema imunológico funcional cardíaca

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Seguindo o destino celular, em<em&gt; E. coli</em&gt; Após a infecção por um fago Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Isolamento de citometria de fluxo de células primárias Murino Tipo II epiteliais alveolares de Estudos funcionais e Molecular

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

Isolamento de tecido adiposo de células imunes

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

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

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

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 ...