Este trabalho foi apoiado pela concessão do NIH P20 GM109098 e pelo Programa de Prêmio de Inovação da Praespero a Edwin Wan. Os experimentos de Citometria de Fluxo foram realizados no WVU Flow Cytometry & Single Cell Core Facility, que é suportado pelas bolsas NIH S10 OD016165, U57 GM104942, P30 GM103488 e P20 GM103434.

Todos os protocolos e procedimentos realizados foram aprovados pelo Comitê Institucional de Cuidados e Uso de Animais (IACUC) da Universidade da Virgínia Ocidental. Os camundongos foram alojados sob condições específicas sem patógenos no vivarium da Universidade da Virgínia Ocidental.
1. Preparação de soluções
<ol><li>Prepare o tampão de perfusão (salina tampão fosfato, PBS). Use aproximadamente duas alíquotas de 10 mL de PBS geladas por mouse.</li>
	<li>Prepare o tampão médio/FACS da célula pulmonar. O tampão FACS contém PBS suplementado com 1% de soro bovino fetal (FBS). Mantenha o meio frio durante todo o processo de excisão pulmonar e transferência. Prepare aproximadamente 8 mL por amostra de pulmão. Prepare um buffer médio/FACS fresco antes dos experimentos.</li>
	<li>Prepare o tampão de dissociação para o isolamento de célula única. O buffer contém 1 mg/mL de colagenase D e 200 μg/mL DNase I na solução de sal tampão da Hank (HBSS). Prepare-se fresco da solução de estoque (100 mg/mL colagenase D e 10 mg/mL DNase I) antes dos experimentos. Aproximadamente 6 mL por amostra de pulmão é necessário. Certifique-se de que o buffer atinja a temperatura ambiente antes do uso.</li>
	<li>Prepare o tampão de homogeneização para homogeneização do tecido pulmonar. O buffer contém PBS e 1x proteinase e coquetel inibidor de fosfatase (estoque = 100x). Prepare-se recentemente antes dos experimentos. Mantenha o tampão frio durante todo o processo de homogeneização. Aproximadamente 200 μL do buffer é suficiente para homogeneizar um único lobo direito dos pulmões.</li>
</ol>2. Oclusão da artéria média transitória (tMCAO)
NOTA: Os procedimentos para tMCAO via inserção de monofilamento na artéria cerebral média foram documentados em detalhes anteriormente10. Nestes experimentos, foram utilizados camundongos C57BL/6J masculinos de 8 a 12 semanas, pesando 25-30 g.
<ol><li>Resumindo, subcutâneamente administre 2 mg/kg de meloxicam nos camundongos antes da cirurgia. Anestesiar profundamente os ratos em uma câmara de indução usando 5% de isoflurane. Confirme a anestesia profunda usando o método de aperto do dedo do dedo do dedo. Mantenha a anestesia com isoflurano de 1 a 2% durante a cirurgia usando um cone no nariz. Mantenha a temperatura corporal em 36,5 - 38 °C durante todo o procedimento, colocando um cobertor quente sob os ratos. Todas as ferramentas cirúrgicas devem ser esterilizadas automaticamente um dia antes da cirurgia.</li>
	<li>Raspe e prepare a pele no pescoço ventral usando 70% de etanol seguido de esfoliante de clorexidina. Administrously administrou 2 mg/kg de bupivacaína sobre a área de incisão antes de fazer a primeira incisão. Use pomada para cobrir os olhos para evitar o ressecamento. Faça uma incisão no pescoço. Puxe suavemente os tecidos moles ao redor da traqueia.</li>
	<li>Identifique a artéria carótida comum esquerda e a artéria carótida externa.</li>
	<li>Aplique uma sutura temporária (tamanho 6/0) na artéria carótida comum para conter o fluxo do sangue. Faça uma incisão na artéria carótida externa.</li>
	<li>Insira um monofilamento revestido de borracha de silicone (tamanho 6-0) na artéria carótida externa e, em seguida, avance o monofilamento para a artéria cerebral média. Deixe o monofilamento na artéria cerebral média por 60 minutos (oclusão).</li>
	<li>Monitore a taxa de fluxo sanguíneo usando laser Doppler Flowmetry. Uma redução da vazão para a artéria cerebral média que é > 80% indica uma oclusão bem sucedida.</li>
	<li>Após os 60 minutos de oclusão, remova o monofilamento para permitir que a reperfusão ocorra. Feche a incisão.</li>
	<li>Em camundongos operados por farsa, realize as etapas 2.1-2.6 sem a inserção do monofilamento. Nestes camundongos, a taxa de fluxo sanguíneo deve permanecer estável durante todo o procedimento.</li>
	<li>Monitore os camundongos diariamente e meça os déficits neurológicos usando critérios de pontuação padrão11. 0 – sem déficit neurológico; 1 – retrai a prenéus contralateral quando levantada pela cauda; 2- círculos para o contralateral quando levantados pela cauda; 3- cair para o contralateral enquanto caminhava; 4 – não anda espontaneamente ou está em coma; 5 – morto.</li>
	<li>Fornecer injeções subcutâneas de soro fisiológico normal e analgésico (meloxicam, 2 mg/kg) a cada 24 horas durante 3 dias após a cirurgia.</li>
	<li>Isole os pulmões 24 e 72 h após a tMCAO para a análise a jusante.</li>
</ol>3. Colhendo os tecidos pulmonares
<ol><li>Anestesia profundamente o camundongo por injeção intraperitoneal (i.p.) de 100 mg/kg de cetamina e 10 mg/kg de xilazina. Confirme a anestesia profunda usando o método de aperto do dedo do dedo do dedo.</li>
	<li>Para realizar toda a perfusão transcárdiado corpo 12, fixe o rato na plataforma cirúrgica e pele molhada com 70% de etanol para reduzir a distribuição de peles na cavidade corporal. Use uma tesoura de dissecção fina para fazer uma incisão transversal no abdômen caudal e, em seguida, uma incisão midline do peritônio, parando na cavidade torácica antes de perfurar o diafragma.<ol><li>Não corte o músculo/se esconda na cavidade peritoneal e não continue a incisão após o abdômen.</li>
	</ol></li>
	<li>Pare a incisão assim que a cavidade torácica for alcançada antes de perfurar o diafragma. Tome cuidado para não danificar o coração e os pulmões que podem comprometer a qualidade da perfusão ou a recuperação celular</li>
	<li>O uso de uma tesoura de dissecção fina faz uma incisão midline do peritônio parar na cavidade torácica antes de perfurar o diafragma.</li>
	<li>Segure o rato pela extremidade distal do esterno usando fórceps e levante para cima enquanto corta o diafragma sendo certo para não danificar o coração, pulmões ou vasculatura. Uma vez que os órgãos são visíveis faça uma incisão através da caixa torácica e separe e fixe a caixa da costela para que o coração e os pulmões sejam totalmente expostos.</li>
	<li>Faça cuidadosamente uma pequena incisão no átrio direito perto do arco aórtico. Isso servirá como saída para a perfusão.</li>
	<li>Imediatamente depois, insira suavemente uma agulha de 25 G x 5/8 presa a uma seringa cheia de 10 mL de PBS frio na superfície superior distal do ventrículo esquerdo. Use cuidado para não inserir através do septo ventricular no ventrículo direito. A agulha mal deve ser inserida no ventrículo esquerdo. NOTA: Se o septo ventricular for perfurado, o fluido sairá correndo do nariz do rato. Isso diminuirá a qualidade da perfusão e resultará em perda celular.</li>
	<li>Se desejar, um grampo pode ser usado para segurar a agulha firmemente no lugar no ventrículo esquerdo durante a perfusão.</li>
	<li>De forma constante, mas suavemente, empurre 10 mL de PBS frio para o coração. O tecido do rato deve começar a perusumar e limpar o sangue à medida que o fluido sai do átrio direito.<ol><li>Empurre uma segunda alíquota de 10 mL de PBS até que tenha ocorrido uma liberação sanguínea suficiente. O fígado terá uma aparência marrom clara e os pulmões passarão de um rosa avermelhado para a cor mais branca.</li>
	</ol></li>
	<li>Usando fórceps e tesouras de dissecção fina, remova cuidadosamente todos os tecidos circundantes, incluindo o coração, traqueia, esôfago, timo, tecido conjuntivo, linfonodos e brônquios grandes.</li>
	<li>Separe os lóbulos pulmonares individuais e coloque os lóbulos em uma placa de Petri no gelo contendo uma alíquota de 1-2 mL de meio de célula pulmonar fria.</li>
</ol>4. Homogeneização do tecido pulmonar para matrizes de contas multiplex usando homogeneizador de contas
<ol><li>Pré-esfrie um tubo de tampa cônica de 2 mL contendo 3 contas estéreis de 2,3 mm de zircônia/sílica. Adicione 200 μL de tampão de homogeneização a frio ao tubo.</li>
	<li>Pesar o lóbulo, transferir o lobo para o tubo cônico pré-refrigerado contendo as contas e o tampão de homogeneização. NOTA: Aproximadamente 50-100 mgs de tecido homogeneizado em 200 μL de tampão serão suficientes para detectar citocinas e expressão de quimiocina na amostra.<ol><li>Homogeneize o lobo usando um homogeneizador à base de contas (ver Tabela de Materiais) a 4.000 rpm por 2 min.</li>
		<li>Certifique-se de que o tecido está completamente homogeneizado. Aumente o tempo, se necessário.</li>
	</ol></li>
	<li>Após a homogeneização, centrifugar o tubo em uma micro centrífuga pré-refrigerada (4 °C) a 15.870 x g por 3 min.</li>
	<li>Transfira os supernantes para um microtubo pré-refrigerado de 1,5 mL.</li>
	<li>Coloque a amostra diretamente no gelo para uso imediato ou armazene a amostra a -80 °C para uso futuro. Evite vários ciclos de congelamento.</li>
</ol>5. Dissociação do tecido pulmonar e isolamento de células únicas
<ol><li>Despeje o meio da célula pulmonar fora dos lóbulos pulmonares restantes para evitar a diluição do tampão de dissociação. Em seguida, insira cuidadosamente uma seringa de 1 mL com agulha 25G e 5/8 contendo 1 mL do tampão de dissociação no tecido pulmonar.</li>
	<li>Injete pequenas frações (aproximadamente 1/4 a 1/5) do tampão de dissociação em cada lobo pulmonar e infle suavemente. NOTA: A maior parte do buffer sairá logo após inflar, pois o lobo foi extirpado.</li>
	<li>Repita a injeção de tampão de dissociação 1-2x.</li>
	<li>Incubar os lóbulos pulmonares com tampão de dissociação por 2 minutos à temperatura ambiente.</li>
	<li>Pique finamente os lóbulos pulmonares em pequenos pedaços usando uma tesoura de dissecção fina.</li>
	<li>Transfira as peças pulmonares picadas com o tampão de dissociação para um tubo centrífuga de 15 mL.  Suplementar com tampão de dissociação adicional para um total de 6 mL. Vórtice vigorosamente por 1 min.</li>
	<li>Incubar a amostra por 45 min a 37 °C. Vórtice vigorosamente a cada 7-8 minutos.</li>
	<li>Após 45 minutos de incubação, vórtice a amostra vigorosamente por 1 min para resultados ideais.</li>
	<li>Dissocia ainda mais a amostra passando por um coador de células de 100 μm para remover tecido conjuntivo residual, indesejado e intersticial.</li>
	<li>Centrifugar a amostra por 10 min a 380 x g, descarte o supernaspe.</li>
	<li>Adicione 5 mL de meio de célula pulmonar fria à amostra. Resuspend a pelota celular com vórtice suave.</li>
	<li>Centrifugar a amostra por 10 min a 380 x g, descarte o supernaspe.</li>
	<li>Adicione 1 mL de meio de célula pulmonar fria. Resuspend a pelota celular com vórtice suave.</li>
	<li>Passe por um coador de células de 100 μm e conte células.</li>
</ol>6. Análise citométrica de fluxo para o nicho de células imunes pulmonares
<ol><li>Semente 1-2 x 106 células/anticorpos definidos em uma placa inferior bem redonda de 96 (3 conjuntos no total). Centrifugar as células por 3 min a 830 x g. Descarte supernantes.</li>
	<li>Resuspenja a pelota de célula em 50 μL de PBS frio contendo mancha viva/morta fixa. Prepare a mancha de acordo com as instruções do fabricante.</li>
	<li>Incubar as células a 4 °C por 20 min protegidas da luz. Centrifugar as células por 3 min a 830 x g. Descarte supernantes.</li>
	<li>Resuspenha a pelota celular com 25 μL de tampão FACS frio contendo 5 μg/mL de anticorpo cd16/32 anti-rato. Incubar a 4 °C por 10-15 min.</li>
	<li>Adicione 25 μL de tampão FACS frio contendo combinações de anticorpos listados na Tabela 1 às células. Misture por pipetação suave. NOTA: O volume total do tampão FACS para a coloração do anticorpo é de 50 μL. Portanto, ao preparar uma mistura mestre de anticorpos em 25 μL, a concentração de anticorpos deve ser o dobro da concentração final.</li>
	<li>Incubar as células a 4 °C por 20 min protegidas da luz. Centrifugar as células por 3 min a 830 x g. Descarte supernantes.</li>
	<li>Resuspenda as células com 100 μL de tampão FACS frio. Centrifugar as células por 3 min a 830 x g. Descarte supernantes.</li>
	<li>Resuspenda as células com 100 μL de tampão FACS frio. Transfira para um tubo de 5 mL FACS de fundo redondo de poliestireno contendo 150 μL de buffer adicional de FACs. Analise a amostra imediatamente usando um citômetro de fluxo. NOTA: A fixação celular usando as seguintes etapas permite que as amostras sejam analisadas posteriormente.</li>
	<li>Seguindo o passo 6.7., resuspenque a amostra com 100 μL de 1% de paraformaldeído na PBS.</li>
	<li>Incubar a amostra a 4 °C por 15 min protegido da luz. Centrifugar as células por 3 min a 830 x g. Descarte supernantes.</li>
	<li>Resuspenda as células com 100 μL de tampão FACS frio. Centrifugar as células por 3 min a 830 x g. Descarte supernantes.</li>
	<li>Resuspenda as células com 100 μL de tampão FACS frio. Armazene as células a 4 °C, protegidas contra a luz. Analise a amostra dentro de 72 h.</li>
</ol>7. Matrizes de contas multiplex para citocina e detecção de quimiocina
NOTA: Os painéis de quimiocina proinflamatória disponíveis comercialmente e os painéis multiplex de inflamação (ver Tabela de Materiais) foram utilizados para determinar a expressão de quimiocinas e citocinas nos pulmões após a indução de tMCAO de acordo com o protocolo do fabricante, que foi descrito no detalhe13.
<ol><li>Em suma, descongele os analitos no gelo se forem armazenados a -80 °C após a homogeneização do tecido. Centrifugar a amostra a 590 x g por 2 min a 4 °C antes de usar para remover qualquer tecido residual.</li>
	<li>Gere uma curva padrão diluindo serialmente o coquetel padrão (C7) que está em uma concentração conhecida de 10 ng/mL em 7 padrões (C1-6) com o último padrão sendo tampão de ensaio sozinho (C0).</li>
	<li>Uma vez que todos os reagentes tenham aquecido à temperatura ambiente, adicione 25 μL de cada padrão e 25 μL de tampão de ensaio a uma placa de poço V inferior 96.</li>
	<li>Para cada amostra bem, adicione 25 μL de homogeneizar pulmonar (analito) e 25 μL de tampão de ensaio a uma placa de poço fundo V 96</li>
	<li>Vórtice as contas pré-revestidas vigorosamente, em seguida, adicionar 25 μL de contas pré-revestidas a cada um dos poços padrão e amostra.</li>
	<li>Sele a placa e proteja da luz cobrindo com papel alumínio. Agite a placa a 110 rpm por 2h em temperatura ambiente.</li>
	<li>Centrifugar a 230 x g por 5 min. Adicione 200 μL de tampão de lavagem em 1x (a solução de estoque é de 20x, diluída a 1x com água). Incubar por 1 min.</li>
	<li>Centrifugar a 230 x g por 5 min. Resuspenda o padrão e a amostra com anticorpos de detecção de 25 μL.</li>
	<li>Veda e cubra a placa para proteger da luz. Agitar a placa a 110 rpm por 1h em temperatura ambiente.</li>
	<li>Adicione 25 μL de streptavidin-phycoerythrin (SA-PE) a cada padrão e prove bem.</li>
	<li>Veda e cubra a placa para proteger da luz. Agite a 110 rpm por 30 min à temperatura ambiente.</li>
	<li>Gire a placa a 230 x g por 5 min. Resuspenda o padrão e as amostras em 1x tampão de lavagem.</li>
	<li>Transfira para um tubo de lavagem de poliestireno bem rotulado de 5 mL FACS contendo um adicional de 150 μL de tampão de lavagem 1x.</li>
	<li>Determine a intensidade de fluorescência do sinal PE para os padrões e amostras usando um citômetro de fluxo.</li>
	<li>Construa uma curva padrão para cada quimiocina e citocina usando a intensidade média de fluorescência (IMC) de PE contra as concentrações pré-determinadas.</li>
	<li>Determine a concentração de cada quimiocina e citocina usando a curva padrão e o MFI de cada amostra. O painel multiplex fornece software de análise de dados que pode ser usado para determinar a concentração de cada analito. O peso do lobo apical/superior é usado para determinar a concentração de citocinas ou quimiocinas por miligrama de tecido.</li>
</ol>

<ol>
	<li>Lloyd, C. M., Marsland, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+Homeostasis:+Influence+of+Age,+Microbes,+and+the+Immune+System.">Lung Homeostasis: Influence of Age, Microbes, and the Immune System.</a> Immunity. 46 (4), 549-561 (2017).</li><li>Allard, B., Panariti, A., Martin, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+Macrophages+in+the+Resolution+of+Inflammation,+Tissue+Repair,+and+Tolerance+to+Infection.">Alveolar Macrophages in the Resolution of Inflammation, Tissue Repair, and Tolerance to Infection.</a> Frontiers in Immunology. 9, 1777 (2018).</li><li>Hartl, 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=Innate+Immunity+of+the+Lung:+From+Basic+Mechanisms+to+Translational+Medicine.">Innate Immunity of the Lung: From Basic Mechanisms to Translational Medicine.</a> Journal of Innate Immunity. 10 (5-6), 487-501 (2018).</li><li>Prass, K., 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=Stroke-induced+immunodeficiency+promotes+spontaneous+bacterial+infections+and+is+mediated+by+sympathetic+activation+reversal+by+poststroke+T+helper+cell+type+1-like+immunostimulation.">Stroke-induced immunodeficiency promotes spontaneous bacterial infections and is mediated by sympathetic activation reversal by poststroke T helper cell type 1-like immunostimulation.</a> Journal of Experimental Medicine. 198 (5), 725-736 (2003).</li><li>Smith, C. 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=Interleukin-1+receptor+antagonist+reverses+stroke-associated+peripheral+immune+suppression.">Interleukin-1 receptor antagonist reverses stroke-associated peripheral immune suppression.</a> Cytokine. 58 (3), 384-389 (2012).</li><li>McCulloch, L., Smith, C. J., McColl, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenergic-mediated+loss+of+splenic+marginal+zone+B+cells+contributes+to+infection+susceptibility+after+stroke.">Adrenergic-mediated loss of splenic marginal zone B cells contributes to infection susceptibility after stroke.</a> Nature Communications. 8 (1), 15051 (2017).</li><li>Dames, 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=Immunomodulatory+treatment+with+systemic+GM-CSF+augments+pulmonary+immune+responses+and+improves+neurological+outcome+after+experimental+stroke.">Immunomodulatory treatment with systemic GM-CSF augments pulmonary immune responses and improves neurological outcome after experimental stroke.</a> Journal of Neuroimmunology. 321, 144-149 (2018).</li><li>Jin, R., Liu, S., Wang, M., Zhong, W., Li, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+CD147+Attenuates+Stroke-Associated+Pneumonia+Through+Modulating+Lung+Immune+Response+in+Mice.">Inhibition of CD147 Attenuates Stroke-Associated Pneumonia Through Modulating Lung Immune Response in Mice.</a> Frontiers in Neurology. 10, 853 (2019).</li><li>Yu, Y. 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=A+Protocol+for+the+Comprehensive+Flow+Cytometric+Analysis+of+Immune+Cells+in+Normal+and+Inflamed+Murine+Non-Lymphoid+Tissues.">A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues.</a> PLoS One. 11 (3), 0150606 (2016).</li><li>Rousselet, E., Kriz, J., Seidah, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+intraluminal+MCAO:+cerebral+infarct+evaluation+by+cresyl+violet+staining.">Mouse model of intraluminal MCAO: cerebral infarct evaluation by cresyl violet staining.</a> Journal of Visualized Experiments. (69), e4038 (2012).</li><li>Farris, B. Y., 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=Ischemic+stroke+alters+immune+cell+niche+and+chemokine+profile+in+mice+independent+of+spontaneous+bacterial+infection.">Ischemic stroke alters immune cell niche and chemokine profile in mice independent of spontaneous bacterial infection.</a> Immunity, Inflammation and Diseases. 7 (4), 326-341 (2019).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments. (65), e3564 (2012).</li><li>Lehmann, J. S., Zhao, A., Sun, B., Jiang, W., Ji, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+Cytokine+Profiling+of+Stimulated+Mouse+Splenocytes+Using+a+Cytometric+Bead-based+Immunoassay+Platform.">Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform.</a> Journal of Visualized Experiments. (129), e56440 (2017).</li><li>Shi, C., Pamer, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+recruitment+during+infection+and+inflammation.">Monocyte recruitment during infection and inflammation.</a> Nature Reviews Immunology. 11 (11), 762-774 (2011).</li><li>Monaghan, K. L., Zheng, W., Hu, G., Wan, E. C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytes+and+Monocyte-Derived+Antigen-Presenting+Cells+Have+Distinct+Gene+Signatures+in+Experimental+Model+of+Multiple+Sclerosis.">Monocytes and Monocyte-Derived Antigen-Presenting Cells Have Distinct Gene Signatures in Experimental Model of Multiple Sclerosis.</a> Frontiers in Immunology. 10, 2779 (2019).</li><li>Zhai, X., 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=A+novel+technique+to+prepare+a+single+cell+suspension+of+isolated+quiescent+human+hepatic+stellate+cells.">A novel technique to prepare a single cell suspension of isolated quiescent human hepatic stellate cells.</a> Science Reports. 9 (1), 12757 (2019).</li><li>Platzer, 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=Dendritic+cell-bound+IgE+functions+to+restrain+allergic+inflammation+at+mucosal+sites.">Dendritic cell-bound IgE functions to restrain allergic inflammation at mucosal sites.</a> Mucosal Immunology. 8 (3), 516-532 (2015).</li><li>Shinoda, K., 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=Thy1+IL-7++lymphatic+endothelial+cells+in+iBALT+provide+a+survival+niche+for+memory+T-helper+cells+in+allergic+airway+inflammation.">Thy1+IL-7+ lymphatic endothelial cells in iBALT provide a survival niche for memory T-helper cells in allergic airway inflammation.</a> Proceedings of the National Academy of Sciences U. S. A. 113 (20), 2842-2851 (2016).</li><li>Nakahashi-Oda, 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=Apoptotic+epithelial+cells+control+the+abundance+of+Treg+cells+at+barrier+surfaces.">Apoptotic epithelial cells control the abundance of Treg cells at barrier surfaces.</a> Nature Immunology. 17 (4), 441-450 (2016).</li><li>Barrott, J. 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=Modeling+synovial+sarcoma+metastasis+in+the+mouse:+PI3'-lipid+signaling+and+inflammation.">Modeling synovial sarcoma metastasis in the mouse: PI3'-lipid signaling and inflammation.</a> Journal of Experimental Medicine. 213 (13), 2989-3005 (2016).</li><li>Bout&#233;, 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+C3HeB/FeJ+mouse+model+recapitulates+the+hallmark+of+bovine+tuberculosis+lung+lesions+following+Mycobacterium+bovis+aerogenous+infection.">The C3HeB/FeJ mouse model recapitulates the hallmark of bovine tuberculosis lung lesions following Mycobacterium bovis aerogenous infection.</a> Veterinary Research. 48 (1), 73 (2017).</li><li>Bal, S. 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=IL-1&#946;,+IL-4+and+IL-12+control+the+fate+of+group+2+innate+lymphoid+cells+in+human+airway+inflammation+in+the+lungs.">IL-1&#946;, IL-4 and IL-12 control the fate of group 2 innate lymphoid cells in human airway inflammation in the lungs.</a> Nature Immunology. 17 (6), 636-645 (2016).</li><li>Nakasone, 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=Accumulation+of+gamma/delta+T+cells+in+the+lungs+and+their+roles+in+neutrophil-mediated+host+defense+against+pneumococcal+infection.">Accumulation of gamma/delta T cells in the lungs and their roles in neutrophil-mediated host defense against pneumococcal infection.</a> Microbes Infection. 9 (3), 251-258 (2007).</li></ol>

Os autores não têm nada a revelar.

Os protocolos aqui descritos permitem a identificação de tipos de células imunes pulmonares e a expressão de quimiocinas ou citocinas no mesmo rato. Se um estudo de histopatologia for desejado, um lobo individual pode ser removido e fixado para esse fim antes de prosseguir para as etapas de isolamento de célula única. Uma limitação deste método é que essa abordagem pode não ser adequada em alguns cenários da doença se a mudança na composição das células imunes e a expressão de quimiocinas e/ou citocinas...

Os pulmões são um órgão de barreira, exposto ao ambiente externo e, portanto, estão constantemente recebendo desafios imunológicos, como patógenos e alérgenos1. A ativação de células imunes residentes no pulmão e a infiltração de células imunes da periferia são necessárias para limpar patógenos do ambiente pulmonar. Além disso, as células imunes residentes no pulmão mantêm a tolerância às bactérias commensais, sugerindo que essas células desempenham um papel no desembaraço do patógeno e na manutenção da homeostase1. Macrófagos alveolares e intersticiais estão entre as células imunes sentinelas residentes no pulmão que sentem patógenos através de receptores de reconhecimento de padrões e limpam esses patógenos por fagocitose2. Células dendríticas residentes no pulmão conectam a resposta imune inata e adaptativa através da apresentação de antígeno3. Além disso, células imunes inatas locais ativadas produzem citocinas e quimioterapias que amplificam a resposta inflamatória e estimulam a infiltração de células imunes como monócitos, neutrófilos e linfócitos nos pulmões1. O AVC isquêmico mostrou-se modificar a imunidade sistêmica e levar ao aumento da suscetibilidade à infecção pulmonar; no entanto, poucos estudos avaliaram o compartimento pulmonar após o acidente vascular cerebral isquêmico, embora alguns estudos o tenham examinado durante as condições inflamatórias 4,5,6,7,8,9. O objetivo dos métodos descritos aqui é determinar simultaneamente a patologia pulmonar, a composição das células imunes e os níveis de citocina e expressão de quimiocina nos pulmões para avaliar alterações no compartimento pulmonar e avaliar possíveis alterações na resposta imune pulmonar após ataque isquêmico.
Descrito aqui é um protocolo para obter suspensões de células únicas dos pulmões dos camundongos para identificar 13 tipos de células imunes. Este protocolo é baseado na digestão tecidual com colagemnase D sem a necessidade de um dissociador de tecido automatizado. Além disso, desenvolvemos um protocolo para preparar homogeneizadores teciduais que podem ser usados para determinar os níveis de expressão de 13 citocinas diferentes ou quimiocinas usando matrizes de contas multiplex baseadas em cytometria de fluxo. Este protocolo foi usado com sucesso para investigar os efeitos do derrame isquêmico na imunidade pulmonar e também pode ser usado em outros modelos de doenças.

<table border="0" cellpadding="0" cellspacing="0" style="width:932px;" width="931">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">B220-APC, clone RA3-6B2</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">103212</td>
			<td style="width:412px;">1:200 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Beadbug 3 position bead homogenizer</td>
			<td style="width:129px;">Benchmark Scientific</td>
			<td style="width:101px;">D1030</td>
			<td>Tissue homogenizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CCR2-BV421, clone SA203G11</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">150605</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD103-BV421, clone 2E7</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">121422</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD11b-PE/Cy7, clone M1/70</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">101216</td>
			<td>1:400 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD11c-PE/Cy7, clone N418</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">117318</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD11c-Percp/Cy5.5, clone N418</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">117328</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD4-BV421, clone GK1.5</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">100443</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD45-FITC, clone 30-F11</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">103108</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD64-APC, clone X54-5/7.1</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">139306</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD8-PE, clone 53-6.7</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">100708</td>
			<td>1:800 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Collagenase D</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td>11088882001</td>
			<td>Component in the dissociation buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Conical screw cap tube</td>
			<td style="width:129px;">ThermoFisher</td>
			<td style="width:101px;">02-681-344</td>
			<td>Tube for tissue homogenization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">DNase I</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td>10104159001</td>
			<td>Component in the dissociation buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Fc block CD16/32 antibody</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">101320</td>
			<td>1:100 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">genlteMACS dissociator</td>
			<td style="width:129px;">Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td>Comparsion of lung digestion with or without mechanical dissociator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">gentleMACS C tubes</td>
			<td style="width:129px;">Miltenyi Biotec</td>
			<td style="width:101px;">130-093-237</td>
			<td>Tube for tissue disscoiation with genlteMACS dissociator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Halt protease and phosphatase inhibitor cocktial</td>
			<td style="width:129px;">ThermoFisher</td>
			<td style="width:101px;">78442</td>
			<td>Component in the homogenization buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Laser doppler monitor</td>
			<td style="width:129px;">Moor</td>
			<td style="width:101px;">MOORVMS-LDF</td>
			<td>Blood flow monitoring during tMCAO</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">LEGENDplex proinflammatory chemokine panel</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">740451</td>
			<td>Multiplex bead array</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">LIVE/DEAD fixable near-IR stain</td>
			<td style="width:129px;">ThermoFisher</td>
			<td style="width:101px;">L34976</td>
			<td>Use for dead cell exclusion during flow cytometric analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Ly6C-PE, clone HK1.4</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">128008</td>
			<td>1:800 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Ly6G-BV510, clone 1A8</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">127633</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">MCAO suture L56 reusable 6-0 medium</td>
			<td style="width:129px;">Doccol</td>
			<td style="width:101px;">602356PK10Re</td>
			<td>tMCAO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">MHC II-BV510, clone M5/114.15.2</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">107636</td>
			<td>1:800 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">NK1.1-Percp/Cy5.5, clone PK136</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">108728</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Siglec F-PE, clone E50-2440</td>
			<td style="width:129px;">BD Biosciences</td>
			<td style="width:101px;">552126</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Silk suture thread, size 6/0</td>
			<td style="width:129px;">Fine Science Tools</td>
			<td style="width:101px;">18020-60</td>
			<td>tMCAO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">SomnoSuite anesthesia system</td>
			<td style="width:129px;">Kent Scientific</td>
			<td style="width:101px;">SS-01</td>
			<td>Mouse anaesthetization for tMCAO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">TCRb-BV510, clone H57-897</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">109234</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Zirconia/silica beads, 2.3 mm</td>
			<td style="width:129px;">Biospec</td>
			<td style="width:101px;">11079125z</td>
			<td>Beads for tissue homogenization</td>
		</tr>
	</tbody>
</table>

characterization of immune cells and proinflammatory mediators in the pulmonary environment

A expansão, ativação e tráfico de células imunes aos pulmões, que são controladas pela expressão de múltiplas citocinas e quimiocinas, podem ser alteradas por lesões cerebrais graves. Isso é evidenciado pelo fato de que a pneumonia é uma das principais causas de mortalidade em pacientes que sofreram de AVC isquêmico. O objetivo deste protocolo é descrever o uso de análises citométricas de fluxo multicolorido para identificar 13 tipos de células imunes nos pulmões de camundongos, incluindo macrófagos alveolares, macrófagos intersticiciais, células dendríticas CD103+ ou CD11b+ (DCs), DCs plasmacitoides, eosinófilos, monocitos/células derivadas de monocitos, neutrófilos, células T e B derivadas de linfoides, células NK e NKT, após indução de derrame isquêmico por oclusão de artéria cerebral média transitória. Além disso, descrevemos a preparação de homogeneizadores pulmonares utilizando um método de homogeneização de contas, para determinar os níveis de expressão de 13 citocinas diferentes ou quimiocinas simultaneamente por matrizes multiplex de contas acopladas à análise citométrica de fluxo. Este protocolo também pode ser usado para investigar a resposta imune pulmonar em outros ambientes da doença, como doença pulmonar infecciosa ou doença alérgica.

Recentemente, relatamos que a indução de derrame isquêmico em camundongos altera a composição celular imune dos pulmões11. Especificamente, isquemia cerebral transitória aumentou percentuais de macrófagos alveolares, neutrófilos e DCs CD11b+, enquanto diminuiu percentuais de células T CD4+, células T CD8+, células B, células NK e eosinófilos no compartimento pulmonar. Além disso, a alteração celular correspondeu a níveis significativamente reduzidos de quimiocinas múltiplas nos ...

Watch this Scientific Journal Video about Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment at JoVE.com

Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment

Este protocolo descreve o uso de citometria de fluxo para identificar as alterações na composição das células imunes, perfil de citocina e perfil de quimiocina no ambiente pulmonar após a oclusão da artéria cerebral média transitória, um modelo murino de derrame isquêmico.

Caracterização de Células Imunes e Mediadores Proinflamatórios no Ambiente Pulmonar

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be ...

characterization-immune-cells-proinflammatory-mediators-pulmonary

Research

JoVE Journal

Immunology and Infection

3.2K visualizações.  West Virginia University. Este protocolo descreve o uso de citometria de fluxo para identificar as alterações na composição das células imunes, perfil de citocina e perfil de quimiocina no ambiente pulmonar após a oclusão da artéria cerebral média transitória, um modelo murino de derrame isquêmico.

Vídeo: Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment

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 ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Imagem intravital da Thymus rato usando 2-Photon Microscopia

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

Diferenciando papéis funcionais da expressão do gene de células imunes e não-imunes na colite Rato por Transplante de Medula Óssea

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

Sistólica do ventrículo direito Medição de Pressão em combinação com colheita de amostras de tecido pulmonar e imunológica em ratos

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

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 ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Investigando os efeitos dos probióticos sobre pneumocócica Colonização Usando um<em&gt; In Vitro</em&gt; Adesão Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Usando RNA-interferência para investigar a imunitário inatas de resposta em macrófagos

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

Isolamento e Caracterização de neutrófilos com propriedades anti-tumorais

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Isolamento, caracterização e exploração funcional da célula de rede gengival Imune

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...