Os autores e este trabalho foram apoiados pelo NIH / NCI conceder # R01CA172138 (a SBE).

Procedimentos envolvendo indivíduos animais foram aprovados pelo Comitê de Cuidado e Uso Institucional Animal (IACUC) da Universidade de Tel-Aviv. 1. Protocolo Rat <ol><li> preparativos <ol><li> Organizar 2 borboleta 21 agulhas G. Opcionalmente, prepare 2 borboleta 21 g agulhas gumes contundentes mediante a apresentação da borda afiada da agulha. </li><li> Esterilizar instrumentos cirúrgicos: 2 pares de tesouras, fórceps embotados-gumes, hemostática, fórceps dente-de tecidos esterilizadas por autoclave a 121 ° C durante pelo menos 30 min no ambiente gravidade (seco). </li><li> Prepare heparinizado PBS (30 unidades / ml) por adição de 30 unidades de heparina por ml de solução salina tamponada com fosfato (PBS 1x). Use a RT. 35 ml é o volume mínimo necessário por animal. </li><li> Transmitir heparinizado PBS para as linhas de bomba peristáltica e as agulhas borboleta para evitar bolhas de ar. </li></ol></li><li> Perfusão dos Pulmões e recolha de MP-leucócitos <ol><li> Eutanásia do animal por uma overdose de 8% de isoflurano. Confirmar eutanásia por cessação de monitorização de respiração. </li><li> Após interrupção da respiração, abra imediatamente as cavidades peritoneal e no peito, usando a tesoura estéreis e pinças dente de tecidos. Realizar uma incisão abdominal na linha média ao longo do abdómen até o processo xifóide. </li><li> Levante o esterno usando a pinça, cuidadosamente cortar a caixa torácica em ambos os lados, sem perfurar qualquer órgão interno ou grandes vasos sanguíneos. Fixe uma pinça hemostática sobre o esterno, e rostralmente dobrar a caixa torácica para expor o complexo cardiopulmonar. </li><li> Inserir uma agulha de borboleta dentro do ventrículo direito do coração, e no prazo de, aproximadamente, um minuto recolher o volume máximo de sangue para uma seringa (~ 6 ml a partir de 250 g de origem animal). </li><li> Prender a veia cava com uma pinça hemostática para evitar trás perfusão do fígado (ver discussão). </li><li> Segure a agulha borboleta dentro do ventrículo direito ao substituir a seringa contendo o sangue com o tubo de saída do pbomba eristaltic. </li><li> Inserir uma segunda agulha borboleta ligada a uma seringa de 5 mL de colheita para o ventrículo esquerdo, evitando a penetração do septo interventricular. </li><li> Ligar a bomba peristáltica a uma velocidade de cerca de 5 ml / min e recolhem suavemente para dentro da seringa os primeiros ml de perfusato que estão contaminados com sangue. Continue até que a cor perfusato muda de escuro para vermelho pálido. (Descartar o perfuste contaminados com sangue). </li><li> Sem abandono da bomba peristáltica, rapidamente substituir a seringa de 5 mL com uma seringa de colheita de 20 ml de colheita e recolher 20 ml de perfusato pulmão empregando uma velocidade mais elevada de perfusão (até 7 ml / min). monitorizar continuamente o fluxo de perfundido para a seringa de recolha, evitando ao mesmo tempo a formação de vácuo. </li><li> Cessar o fluxo da bomba peristáltica. </li></ol></li><li> leucócitos Extração <ol><li> Centrifuga-se o perfusado durante 10 min a 400 x g. </li><li> Aspirar o fluido. </li><li>Adicionar 10 ml de PBS ou meio, centrifugar a 400 xg durante 10 min, e aspirar o fluido. Repita esta etapa duas vezes. O uso de PBS ou meio depende da consequente utilização da amostra. </li><li> Adicionar 20 ml de PBS ou meio, centrifugar a 400 xg durante 10 min, e aspirar o fluido. </li><li> Reconstituir as células na concentração desejada, com base na utilização da amostra (FACS, PCR, etc.). </li></ol></li></ol> 2. Rato Protocolo <ol><li> preparativos <ol><li> Organizar 2 borboleta 25 agulhas G. Opcionalmente, prepare 2 borboleta 25 g agulhas gumes contundentes mediante a apresentação da borda afiada da agulha. </li><li> Esterilizar instrumentos cirúrgicos: 2 pares de tesouras, fórceps embotados-gumes, hemostática, fórceps dente-de tecidos esterilizadas por autoclave a 121 ° C durante pelo menos 30 min no ambiente gravidade (seco). </li><li> Prepare heparinizado PBS (30 unidades / ml) por adição de 30 unidades de heparina por ml de solução de PBS 1x. Use a RT. 25 ml é o volume mínimo necessário por animal. </li><li> streestou heparinizado PBS para as linhas de bomba peristáltica e as agulhas borboleta para evitar bolhas de ar. </li></ol></li><li> Perfusão dos Pulmões e recolha de MP-leucócitos <ol><li> Eutanásia do animal por uma overdose de 8% de isoflurano. Confirmar eutanásia por cessação de monitorização de respiração. </li><li> Após interrupção da respiração, abra imediatamente as cavidades peritoneal e no peito, usando a tesoura estéreis e pinças dente de tecidos. Realizar uma incisão abdominal na linha média ao longo do abdómen até o processo xifóide. </li><li> Levante o esterno usando a pinça, cuidadosamente cortar a caixa torácica em ambos os lados, sem perfurar qualquer órgão interno ou grandes vasos sanguíneos. Fixe uma pinça hemostática sobre o esterno, e rostralmente dobrar a caixa torácica para expor o complexo cardiopulmonar. </li><li> Prender a veia cava com uma pinça hemostática para evitar trás perfusão do fígado (ver discussão). </li><li> Inserir uma agulha borboleta ligado ao tubo de saída da bomba peristáltica para a direita ventricle do coração, e uma segunda agulha borboleta ligada a uma seringa de 2 ml de colheita para o ventrículo esquerdo do coração, avoidingpenetration do septo interventricular. </li><li> Ligue a bomba a uma velocidade de aproximadamente 2 ml / min e recolher os primeiros ml de perfusato contaminados com sangue na seringa a colheita até que o perfusato muda de escuro para vermelho pálido (Descartar o perfuste contaminados com sangue). </li><li> Sem abandono da bomba peristáltica, rapidamente substituir a 2 ml de seringa de colheita com uma seringa de 10 ml de colheita e recolher 10 ml de perfusato pulmão empregando uma velocidade mais elevada de perfusão (até 4 ml / min). monitorizar continuamente o fluxo de perfundido para a seringa de recolha, evitando ao mesmo tempo a formação de vácuo. </li><li> Cessar o fluxo da bomba peristáltica. </li></ol></li><li> leucócitos Extração <ol><li> Centrifuga-se o perfusado durante 10 min a 400 x g. </li><li> Aspirar o fluido. </li><li> Adicionar 10 ml de PBS ou meio, centrifugar a 400xg durante 10 min, e aspirar o fluido. Repita este passo três vezes. O uso de PBS ou meio depende da consequente utilização da amostra. </li><li> Adicionar 10 ml de PBS ou meio, centrifugar a 400 xg durante 10 min, e aspirar o fluido. </li><li> Reconstituir as células na concentração desejada, com base na utilização da amostra (FACS, PCR, etc.). </li></ol></li></ol>

<ol>
	<li>Kuebler, W. M., Goetz, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marginated+pool.">The marginated pool.</a> Eur Surg Res. 34, 92-100 (2002).</li><li>Benish, M., 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=The+marginating-pulmonary+immune+compartment+in+mice+exhibits+increased+NK+cytotoxicity+and+unique+cellular+characteristics.">The marginating-pulmonary immune compartment in mice exhibits increased NK cytotoxicity and unique cellular characteristics.</a> Immunol Res. 58, 28-39 (2014).</li><li>Melamed, R., 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=The+marginating-pulmonary+immune+compartment+in+rats:+characteristics+of+continuous+inflammation+and+activated+NK+cells.">The marginating-pulmonary immune compartment in rats: characteristics of continuous inflammation and activated NK cells.</a> J Immunother. 33, 16-29 (2010).</li><li>Shakhar, G., 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=Amelioration+of+operation-induced+suppression+of+marginating+pulmonary+NK+activity+using+poly+IC:+a+potential+approach+to+reduce+postoperative+metastasis.">Amelioration of operation-induced suppression of marginating pulmonary NK activity using poly IC: a potential approach to reduce postoperative metastasis.</a> Ann Surg Oncol. 14, 841-852 (2007).</li><li>Smyth, M. J., Godfrey, D. I., Trapani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fresh+look+at+tumor+immunosurveillance+and+immunotherapy.">A fresh look at tumor immunosurveillance and immunotherapy.</a> Nat Immunol. 2, 293-299 (2001).</li><li>Talmadge, J. E., Meyers, K. M., Prieur, D. J., Starkey, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+NK+cells+in+tumour+growth+and+metastasis+in+beige+mice.">Role of NK cells in tumour growth and metastasis in beige mice.</a> Nature. 284, 622-624 (1980).</li><li>Grimm, E. A., Mazumder, A., Zhang, H. Z., Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphokine-activated+killer+cell+phenomenon.+Lysis+of+natural+killer-resistant+fresh+solid+tumor+cells+by+interleukin+2-activated+autologous+human+peripheral+blood+lymphocytes.">Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes.</a> J Exp Med. 155, 1823-1841 (1982).</li><li>Melamed, R., 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=Marginating+pulmonary-NK+activity+and+resistance+to+experimental+tumor+metastasis:+suppression+by+surgery+and+the+prophylactic+use+of+a+beta-adrenergic+antagonist+and+a+prostaglandin+synthesis+inhibitor.">Marginating pulmonary-NK activity and resistance to experimental tumor metastasis: suppression by surgery and the prophylactic use of a beta-adrenergic antagonist and a prostaglandin synthesis inhibitor.</a> Brain Behav Immun. 19, 114-126 (2005).</li><li>O'Dea, K. P., 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=Lung-marginated+monocytes+modulate+pulmonary+microvascular+injury+during+early+endotoxemia.">Lung-marginated monocytes modulate pulmonary microvascular injury during early endotoxemia.</a> Am J Respir Crit Care Med. 172, 1119-1127 (2005).</li><li>Sorski, L., Melamed, R., Ben-Eliyahu, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+role+for+marginating-pulmonary+and+marginating-hepatic+NK+cells+in+cancer+anti-metastatic+surveillance+and+in+immunotherapy.">A unique role for marginating-pulmonary and marginating-hepatic NK cells in cancer anti-metastatic surveillance and in immunotherapy.</a> , (2014).</li><li>Levi, 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=Continuous+stress+disrupts+immunostimulatory+effects+of+IL-12).">Continuous stress disrupts immunostimulatory effects of IL-12).</a> Brain Behav Immun. 25, 727-735 (2011).</li></ol>

Os autores não têm nada a revelar.

O método aqui apresentado permite a colheita selectiva e estudo da população única de MP-leucócitos. Em comparação com circulante ou leucócitos esplénicas, a população MP é caracterizada por uma composição distinta de subpopulações de leucócitos, níveis de activação mais elevadas, maior libertação de várias citocinas, e mais elevados os níveis de mRNA de citocinas pró- e anti-inflamatórios 2,3. Especificamente, mostraram que as células NK-MP são mais citotóxicos do que as células...

Os leucócitos aderentes para os capilares dos pulmões (isto é, marginating-pulmonar (MP) leucócitos) foram 1 mostrado exibir composição distinta de leucócitos e a actividade única em comparação com os leucócitos a partir de outros compartimentos do sistema imunológico (por exemplo, circulação, do baço, da medula óssea) 2- 4. Por exemplo, exposição de MP-leucócitos maior células assassinas naturais (NK) a citotoxicidade contra diversas células tumorais, em comparação com células circulantes e esplénicos NK, bem como os níveis de RNA mensageiro (mRNA) diferenciadas e aumento da secreção de citoquinas pró-inflamatórias e anti. A composição das células também é diferenciado a partir de leucócitos circulantes como MP-leucócitos têm uma proporção mais elevada de imunidade inata / adaptativa em comparação com os leucócitos circulantes (50% vs. 30%, respectivamente). O objectivo do método aqui apresentado é o de permitir a colheita selectiva de MP-leucócitos, a fim de estudar este compartimento imunológica importante e original (população de células), e a elucIdate o impacto das diversas manipulações (por exemplo, ativação imune) sobre essas células específicas. Para compreender o significado deste população original, é importante notar que o sistema imune pode controlar células tumorais, micrometástases e de doença residual que circula através de funções in vivo de imunidade mediada por células (CMI). Esta capacidade é evidente, apesar da falha precedente do sistema imunitário para controlar o tumor primário, e suportado por um amplo evidências in vivo em doentes com cancro e em modelos animais 5. Mais importante, estes resultados são muitas vezes inconsistentes com estudos in vitro e ex vivo, que reportam que a maioria das células tumorais autólogas são resistentes a citotoxicidade por leucócitos em amostras de sangue em circulação a partir de seres humanos e animais (medido por ensaios de citotoxicidade) 6,7. Esta discrepância pode ser atribuída à existência in vivo, de populações de leucócitos distintas, tais como o supramencionado população de leucócitos MP e, especificamente, a sua subpopulação de células NK activadas 3. Com efeito, as células singeneicas de tumor (MADB106), que se verificou ser resistente à circulante e leucócitos esplénicas, foram mostrados para serem lisadas por células NK-MP 3,8. Assim, as células &#39;&#39; resistentes a NK-alegadamente MADB106 que metastizam para os pulmões de ratos fischer344 (F344) são controlados por células NK-MP, mas não por células NK circulantes ou esplénicas, que são vulgarmente estudada, dada a sua facilidade de acesso. Purificada e activas MP-leucócitos são inacessíveis através dos métodos de colheita padrão de leucócitos a partir dos pulmões, os quais são baseados na moedura de tecido pulmonar ou degradação biológica 9. A nossa abordagem tem duas vantagens principais em comparação com abordagens de processamento de tecidos. Em primeiro lugar, a abordagem de perfusão colhe seletivamente MP-leucócitos, separando-os de outras células que se originam a partir do parênquima pulmonar, intersticial e compar bronco-alveolartimentos. Em segundo lugar, a técnica de perfusão melhor preserva a integridade e o meio fisiológico de MP-leucócitos, ao contrário da moagem e processamento biológico abordagens que danificam as células, alteram a sua morfologia, e induzem a produção e libertação de vários factores que modulam a actividade imunológica e especificamente suprimem NK citotoxicidade 10. Os pulmões são um dos principais alvos de metástase do câncer e para várias doenças infecciosas. Todas as células malignas circulantes e células infectadas passar através dos capilares pulmonares, onde eles precisam deformar e interagir com as células endoteliais capilares e leucócitos residentes. Sob estas condições, as células circulantes podem ser facilmente alvo de MP-leucócitos residentes. Parece, portanto, biologicamente vantajoso ter leucócitos activados neste compartimento imune, e é importante para estudar esta MP-população única em diferentes configurações biológicos, experimentais, e clínicos. É digno de nota que syactivação stemic imune por vários-resposta-modificadores biológicos (por exemplo, ácido polyinosinic-polycytidylic (poli I: C) ou oligodesoxinucleótidos tipo-C CpG (CpG-C ODN)) ter mostrado activar MP-leucócitos mais de leucócitos circulantes 3, 8,11.

<table><tbody><tr><td>Autoclud Peristaltic pump</td><td></td><td></td><td></td></tr><tr><td>Butterfly needle</td><td>OMG</td><td></td><td>26G</td></tr><tr><td>Butterfly needle</td><td>OMG</td><td></td><td>21G*3/4&quot;</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>1 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>2.5 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>5 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>10 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>25 ml</td></tr><tr><td>Blunted-edged forceps</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>hemostat</td><td></td><td></td><td></td></tr><tr><td>Tissue forceps</td><td></td><td></td><td></td></tr><tr><td>Heparin sodium porcine mucosa (perservative free)</td><td>Sigma Aldrich</td><td></td><td></td></tr></tbody></table>

selective harvesting of marginating-pulmonary leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

A exposição MP-compartimento uma composição de leucócitos subconjunto diferente em comparação com leucócitos circulantes. Usando a análise de citometria de fluxo, as subpopulações de leucócitos foram identificados e quantificados para caracterizar a composição de ambas as circulante e leucócitos MP. Granulócitos e linfócitos foram identificados com base na frente eo lado espalha. Dentro dos linfócitos, NKRP-1 células vivas foram identificadas como células N...

Watch this Scientific Journal Video about Selective Harvesting of Marginating-pulmonary Leukocytes at JoVE.com

Selective Harvesting of Marginating-pulmonary Leukocytes

Colheita seletiva de Marginating-pulmonar Leucócitos

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

selective-harvesting-of-marginating-pulmonary-leukocytes

Research

JoVE Journal

Immunology and Infection

5.8K visualizações.  Tel-Aviv University. Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Vídeo: Selective Harvesting of Marginating-pulmonary Leukocytes

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Isolamento de células dendríticas pulmonares do rato

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Imunologia e Infecção

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Colheita seletiva de Marginating-hepática Leucócitos

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource to evaluate a variety of pulmonary diseases, both for research and clinical purposes. However, due to the heterogeneous and viscous nature of patient mucus, there is currently no reliable dissociation method that does not damage the host immune cells in the patient airway secretion. In this research, we introduce a sample preparation method that uses inertial microfluidics for the patient&#39;s immune assessment. Regardless of the heterogeneous fluidic properties of the clinical samples, the proposed method recovers more than 95% of neutrophils from airway secretion samples that are diluted 1,000-fold with milliliters of clean saline. By recirculating the concentrated output stream to the initial sample reservoir, a high concentration, recovery, and purity of the immune cells are provided; recirculation is considered a trade-off to the single-run syringe-based operation of inertial microfluidics. The closed-loop operation of spiral microfluidics provides leukocytes without physical or chemical disturbance, as demonstrated by the phorbol 12-myristate 13-acetate (PMA)-induced elastase release of sorted neutrophils.

In this research, we demonstrate a label-free neutrophil separation method from clinical airway secretions using closed-loop operation of spiral inertial microfluidics. The proposed method would expand the clinical in vitro assays for various respiratory diseases.

Livre de rótulo neutrófilo enriquecimento de secreção do paciente-derivado das vias aéreas usando microfluídica inercial de circuito fechado

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource ...

Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach

Circadian rhythms refer to oscillations in various biological process that occur with a 24 h period. At the molecular level, such rhythms are comprised of a web of transcriptional-translational feedback loops (TTFL) of core clock genes. Individual tissues and organ systems, including the immune system, have their own clock. In the systemic circulation, various members of the CD45+ population oscillate across the day; however, many of these rhythms are not identical or even similar in the tissue resident CD45+ leukocyte population. When studying the role of circadian regulation of lung inflammation, CD45+ within the lung may need to be investigated. However, despite optimized perfusion methods, leukocytes trapped from the circulation persist in the lungs. The goal in designing this protocol was to distinguish between intravascular and intraparenchymal leukocytes. Towards this end, mice are injected with a fluorescent tagged CD45 antibody intrajugularly shortly before lung harvest. Thereafter, the lung is digested using a customized lung digestion technique to obtain a single cell suspension. The sample is stained for the regular panel of antibodies for intraparenchymal immune cells (including another CD45 antibody). Flowcytometric analyses shows a clear elucidation of the populations. Thus, the method of labeling and defining intrapulmonary CD45+ cells will be particularly important where the behavior of intrapulmonary and circulating immune cells are numerically and functionally distinct.

The aim of the current study is to describe a protocol for differentiating between intravascular and intraparenchymal immune cells in studies of lung inflammation. We use an intrajugular injection of a fluorescent tagged antibody prior to lung harvest. Further, we use an inflation-based lung digestion process to improve the yield of leukocytes from the lung.

Circadian rhythms refer to oscillations in various biological process that occur with a 24 h period. At the molecular level, such rhythms are comprised of ...

Harvest of Endothelial Cells from the Balloon Tips of Swan-Ganz Catheters after Right Heart Catheterization

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further diagnose and manage PH, patients undergo repeated right heart catheterizations (RHC) wherein a Swan-Ganz catheter is advanced into a branch of the pulmonary artery and a balloon is inflated to wedge the catheter tip. This article illustrates a protocol whereby pulmonary artery endothelial cells (PAECs) may be harvested from the balloon tips of Swan-Ganz catheters after RHC, and purified with an anti- CD146 affinity column technique to purify putative PAECs. These cells might be used to provide an in situ snapshot of the biological state of the pulmonary vasculature endothelium to complement hemodynamic measurements obtained during RHC. Harvested and purified PAECs may be used for either cell culture or for subsequent analytical assays such as flow cytometery.

This protocol describes the removal and purification of pulmonary artery endothelial cells (PAECs) that adhere to the balloon tips of Swan-Ganz catheters while they are wedged in the pulmonary artery during right heart catheterization and remain adherent to the deflated balloon following removal from the patient's body.

Colheita de células endoteliais das dicas de balão de cateteres de Swan-Ganz após cateterismo cardíaco direito

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further ...

Medicina

Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while suppressing unwanted reactions to environmental antigens. Lungs host several populations of innate and adaptive immune cells that provide immune surveillance but also mediate protective immune responses. These cells, which keep the healthy pulmonary immune system in balance, also participate in several pathological conditions such as asthma, infections, autoimmune diseases, and cancer. Selective expression of surface and intracellular proteins provides unique immunophenotypic properties to the immune cells of the lung. Consequently, flow cytometry has an instrumental role in the identification of such cell populations during steady-state and pathological conditions. This paper presents a protocol that describes a consistent and reproducible method to identify the immune cells that reside in the lungs of healthy mice under steady-state conditions. However, this protocol can also be used to identify changes in these cell populations in various disease models to help identify disease-specific changes in the lung immune landscape.

In this study, we present an effective and reproducible protocol to isolate the immune populations of the murine respiratory system. We also provide a method for the identification of all innate and adaptive immune cells that reside in the lungs of healthy mice, using a 9-color-based flow cytometry panel.

Análise Citométrica de Fluxo para Identificação das Células Imunes Inatas e Adaptativas do Pulmão murino

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while ...

<meta charset="utf8"></head><body>Isolamento de células imunes e não imunes de pulmões de camundongos para citometria de fluxo

Isolation of Live Myeloid and Epithelial Cell Populations from the Mouse Lung

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development of disease. Studies utilizing mouse models of respiratory infection, allergy, fibrosis, and cancer have been critical to reveal mechanisms of disease progression and identify therapeutic targets. However, most studies focused on the mouse lung prioritize the isolation of either immune cells or epithelial cells, rather than both populations concurrently. Here, we describe a method for preparing a comprehensive single-cell suspension of both immune and non-immune populations suitable for flow cytometry and fluorescence-activated cell sorting. These populations include epithelial cells, endothelial cells, fibroblasts, and a variety of myeloid cell subsets. This protocol entails bronchoalveolar lavage and subsequent inflation of the lungs with dispase. Lungs are then digested in a liberase mixture. This method of processing liberates a variety of diverse cell types and results in a single-cell suspension that does not require manual dissociation against a filter, promoting cell survival and yielding high numbers of live cells for downstream analyses. In this protocol, we also define gating schemes for epithelial and myeloid cell subsets in both na&#239;ve and influenza-infected lungs. Simultaneous isolation of live immune and non-immune cells is key for interrogating intercellular crosstalk and gaining a deeper understanding of lung biology in health and disease.

<meta charset="utf8"></head><body>Isolamento de populações de células mieloides e epiteliais vivas do pulmão de camundongo

This protocol details the isolation of live immune and non-immune populations from the mouse lung at a steady state and following influenza infection. It also provides gating strategies for identifying epithelial and myeloid cell subsets.

Isolamento de populações de células mieloides e epiteliais vivas do pulmão de camundongo

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development ...

Estabelecimento de culturas LLEC in vitro usando seleção de células magnéticas

Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct from peripheral lymphatic endothelial cells. Their cellular function and clinical implications remain largely unknown. Consequently, the availability of a supply of LLECs is essential for conducting functional research in vitro. However, there is currently no existing protocol for harvesting and culturing LLECs in vitro.
This study successfully harvested LLECs using a multi-step protocol, which included coating the flask with fibronectin, dissecting the leptomeninges with the assistance of a microscope, enzymatically digesting the leptomeninges to prepare a single-cell suspension, inducing the expansion of LLECs with vascular endothelial growth factor-C (VEGF-C), and selecting lymphatic vessel hyaluronic receptor-1 (LYVE-1) positive cells through magnetic-activated cell sorting (MACS). This process ultimately led to the establishment of a primary culture. The purity of the LLECs was confirmed through immunofluorescence staining and flow cytometric analysis, with a purity level exceeding 95%. This multi-step protocol has demonstrated reproducibility and feasibility, which will greatly facilitate the exploration of the cellular function and clinical implications of LLECs.

Autor em destaque: Revelando funções celulares e potenciais implicações clínicas das células endoteliais linfáticas leptomeníngeas 

Leptomeningeal lymphatic endothelial cells (LLECs), a recently identified intracranial cell type, have poorly understood functions. This study presents a reproducible protocol for harvesting LLECs from mice and establishing in vitro primary cultures. This protocol is designed to enable researchers to delve into the cellular functions and potential clinical implications of LLECs.

Colheita e Cultura Primária de Células Endoteliais Linfáticas Leptomeníngeas

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct ...

Colheita seletiva de Marginating-pulmonar Leucócitos

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