Agradecemos a Clare Prendergast para obter assistência com o manuscrito de edição. DKN é suportado por uma pesquisa conceder (#2095) da Fundação Flinn e TM é suportada por concessões do National Institutes of Health (R01HL056643 e R01HL092514). DKN desenvolveu os métodos, projetou o estudo e escreveu o manuscrito; OM assistida com estudos em animais e recolha de amostra clínica; SB assistida com fluxo cytometric análise e classificação de célula; TM supervisionou os estudos e revisão do manuscrito.

Todos os métodos descritos aqui foram aprovados pelo Comitê de uso (IACUC) o cuidados de Animal institucional e o institucional Review Board (IRB), no Hospital e centro médico de St. Joseph.
1. isolamento de AMs do líquido de lavagem broncoalveolar murino (BAL)
<ol><li>Anestesiar um rato C57BL/6 de oito semanas de idade com cetamina (87,5 mg/kg de peso) e xilazina (12,5 mg/kg de peso corporal) coquetel através de uma injeção intraperitoneal. Proceda quando rato atinja a anestesia cirúrgica com perda de reflexos e relaxamento dos músculos.</li>
	<li>Coloque o mouse sobre uma superfície de dissecação com o lado ventral virada para cima. Aplica a pomada oftálmica veterinário nos olhos para evitar a desidratação e limpe toda a superfície ventral do mouse com almofadas de preparação de álcool estéril saturadas com isopropanol 70% para desinfetar.</li>
	<li>Monte o mouse com as quatro patas contidas.</li>
	<li>Abra cuidadosamente a cavidade abdominal com ferramentas micro dissecação. Deve ter cuidado para abrir a área tanto quanto possível sem danificar qualquer órgãos viscerais ou criando tecido pendendo sobre.</li>
	<li>Abra cuidadosamente a cavidade torácica por incisão lentamente a cavidade torácica através do mediastino. Idealmente, a caixa torácica pode ser extirpada do lado a lado. Extremo cuidado deve ser tomado para não machucar a pleura, dos pulmões durante a abertura da cavidade torácica.</li>
	<li>Localizar a veia cava inferior e fazer uma incisão para sangrar o mouse. Use almofadas de algodão absorvente para absorver o sangue.</li>
	<li>Injete soro fisiológico 10ml gelada tamponado de fosfato (PBS) no ventrículo direito (usando a seringa de 10 mL e agulha 25g) para liberar as células do sangue circulantes. Absorva o fluxo de sangue com almofadas de algodão absorvente. Uma vez completamente perfundidos, os pulmões aparecerão branqueados e então estão prontos para lavagem.</li>
	<li>Delicadamente abri a pele e muscular no pescoço para expor as vias aéreas. Excisar cuidadosamente os músculos adjacentes, cartilagens e tecidos de gordura sem danificar a traqueia.</li>
	<li>Faça uma pequena incisão (< 2 mm) na traqueia posterior na laringe. Esta incisão deve ser apenas o suficiente para inserir um cateter em enquanto a traqueia continua preso na laringe. Inserir um cateter de 22G 1 polegada sem uma agulha na traqueia em direção aos pulmões. Fixar o cateter com uma sutura trançada seda (4-0; não absorvível) com um nó quadrado. Nota: O cateter precisa ser aparado com base no tamanho do mouse. Cateteres de extremamente curtos ou longos tendem a vazar e/ou rasgar a via aérea durante a lavagem. Comprimento do cateter deve ser tal que, quando totalmente inseridos os restos de ponta bem dentro da traqueia e não atingir os brônquios primários.</li>
	<li>Anexar uma seringa de 1 mL, cheia de buffer de BAL gelado (Ca2 + e Mg2 + grátis PBS + 1 mM de ácido etilenodiaminotetracético, EDTA) para o cateter e incutir lentamente o buffer para os pulmões. Isto irá inflar os pulmões. Manter a seringa anexada ao cateter por cinco segundos e então aspirar o fluido de lavagem puxando suavemente o êmbolo. Isso esvaziará os pulmões. Recolha o líquido em um tubo cônico de 15 mL colocado no gelo.</li>
	<li>Repita a etapa 1.10 para nove vezes mais e o fluido de lavagem da piscina. Não exceda 1 mL de tampão por descarga, que podem exceder a capacidade pulmonar e levar a sua ruptura.</li>
	<li>Eutanásia o mouse com overdose de CO2 por protocolo IACUC aprovado.</li>
	<li>Centrifugue o líquido 10ml BAL a 250 x g a 4 ° C durante 10 minutos. O centrifugado contém células do BAL. Atenção: O sedimento pode ser muito pequeno, então tenha cuidado ao aspirar o sobrenadante.</li>
	<li>Ressuspender as células do BAL em 100 μL de tampão (PBS + 2% calor inativada soro fetal bovino (FBS) + 1 mM EDTA e ácido N-2-Hydroxyethylpiperazine-N'-2-ethanesulphonic (HEPES) de 25 mM) de fluxo/classificação.</li>
	<li>Bloquear a ligação de anticorpos inespecíficos ao receptor Fc adicionando bloco de Fc de rato (clone 2.4G2, 10 μg/mL) e incubar durante 20 minutos no gelo.</li>
	<li>Executar o anticorpo que mancha com fluorescência menos um (FMO) e single-mancha controles11,25. Avaliar o BAL células por um analisador de célula de análise de citometria de fluxo de célula única (ver Tabela de materiais) seguiram pelo software de análise adequada (consulte a Tabela de materiais).</li>
	<li>Isolar a AMs por uma célula de fluorescência-ativado classificador (ver Tabela de materiais) diretamente em rato 1 mL estou meio de cultura quando AMs destinam-se a cultura in vitro ou no vivo transferência de célula. Como alternativa, tipo AMs em 1 mL de tampão de lise (ver Tabela de materiais) suplementado com 10 µ l/mL de β-Mercaptoetanol para extração de RNA.</li>
</ol>2. isolamento de AMs do pulmão do rato, suspensão de célula única
<ol><li>Anestesiar um rato C57BL/6 de oito semanas de idade com cetamina (87,5 mg/kg de peso) e xilazina (12,5 mg/kg de peso corporal) coquetel através de uma injeção intraperitoneal. Proceda quando rato atinja a anestesia cirúrgica com perda de reflexos e relaxamento dos músculos.</li>
	<li>Coloque o mouse sobre uma superfície de dissecação com o lado ventral virada para cima. Aplica a pomada oftálmica veterinário nos olhos para evitar a desidratação e limpe toda a superfície ventral do mouse com álcool estéril almofadas saturadas com isopropanol 70% para desinfetar.</li>
	<li>Monte o mouse com as quatro patas contidas.</li>
	<li>Abra cuidadosamente a cavidade abdominal com ferramentas micro dissecação. Deve ter cuidado para abrir a área tanto quanto possível sem danificar qualquer órgãos viscerais ou criando tecido pendendo sobre.</li>
	<li>Abra cuidadosamente a cavidade torácica por incisão lentamente a cavidade torácica através do mediastino. Idealmente, a caixa torácica pode ser extirpada do lado a lado. Extremo cuidado deve ser tomado para não machucar a pleura, dos pulmões durante a abertura da cavidade torácica.</li>
	<li>Localizar a veia cava inferior e fazer uma incisão para sangrar o mouse. Use almofadas de algodão absorvente para absorver o sangue.</li>
	<li>Injete 10 mL PBS gelado no ventrículo direito (usando a seringa de 10 mL e agulha 25g) para liberar as células do sangue circulantes. Uma vez completamente perfundidos, os pulmões aparecerão branqueados e estão prontos para a colheita.</li>
	<li>Dissecar os pulmões pelo rompimento da traqueia, vasos sanguíneos e ligamentos em um tubo cônico de 15 mL, com 5 mL de frio médio modificado águia de Dulbecco (DMEM). Mantê-lo no gelo até o próximo passo.</li>
	<li>Eutanásia o mouse com overdose de CO2 por protocolo IACUC aprovado.</li>
	<li>Transferi os pulmões perfundidos para um prato de cultura estéril e apirógena 60 mm com 3 mL DMEM. Com a ajuda de pinças de dissecação destrinchar as vias aéreas e outro material duro tecido não-pulmonar, se presente. Picar o tecido pulmonar com um bisturi para < tamanho de 1 mm.</li>
	<li>Adicionar 300 µ g/mL Liberase TL e 5 U/mL DNase, misturar pipetando e incubar a 37° C numa incubadora por 25 minutos. Misture suavemente uma vez pipetando após 10 minutos.</li>
	<li>Passe os pulmões dissociados através de um filtro de célula 100 µm instalado em um tubo cónico de 50 mL. Use a volta de um êmbolo de seringa de 1 mL para amassar até grupos de célula no filtro. Lavar o filtro com 20 mL de tampão de lavagem (PBS + 2% de calor inactivada FBS + 2 mM EDTA).</li>
	<li>Centrifugar a 500 x g a 4 ° C durante 5 minutos. Desprezar o sobrenadante e ressuspender as células em 20 mL de tampão de lavagem.</li>
	<li>Repita as etapas 2.12 e 2.13 duas vezes, mudando o filtro e tubo de cada vez. Pré-imersão o filtro em tampão de lavagem por mais de cinco minutos aumenta o rendimento de AM. Preparar a suspensão de célula única adicionando 100 μL de tampão para o centrifugado de fluxo/classificação.</li>
	<li>Bloquear a ligação de anticorpos inespecíficos ao receptor Fc adicionando bloco de Fc de rato (clone 2.4G2, 10 μg/mL) e incubar durante 20 minutos no gelo.</li>
	<li>Execute o anticorpo que mancha junto com FMO e controles de single-mancha11,25. Avaliar o pulmão células por um analisador de célula (ver Tabela de materiais) seguiram por citometria de fluxo de célula única análise pelo software.</li>
	<li>AMs tipo por um classificador de pilha (ver Tabela de materiais) diretamente em rato 1 mL estou meio de cultura quando AMs destinam-se a cultura in vitro ou no vivo transferência de célula. Alternativamente, classificar AMs em 1 mL de tampão de Lise suplementado com 10 µ l/mL de β-Mercaptoetanol para extração de RNA.</li>
</ol>3. isolamento de AMs de fluido humano BAL
<ol><li>Transferir o líquido de BAL humano da clínica para o laboratório a 4 ° C.</li>
	<li>Centrifugue o fluido BAL (10-50 mL) em 250 x g a 4 ° C durante 10 minutos. O centrifugado contém BAL células11.</li>
	<li>Lave o pellet com 20 mL de tampão de lavagem (PBS + 2% de calor inactivada FBS + 2 mM EDTA) e centrifugar 250 x g a 4 ° C por 10 minutos.</li>
	<li>Ressuspender as células do BAL estão em 100 μL de tampão de fluxo/classificação (PBS + 2% de calor inactivada FBS + 1 mM EDTA + 25mm HEPES).</li>
	<li>Bloquear a ligação de anticorpos inespecíficos ao receptor Fc adicionando humana Fc bloco (clone Fc1.3070, 25 μg/mL) e incubar durante 20 minutos no gelo.</li>
	<li>Execute o anticorpo que mancha junto com FMO e controles de single-mancha11,24. Avaliar o BAL células por um analisador de célula (ver Tabela de materiais) seguiram por citometria de fluxo de célula única análise pelo software.</li>
	<li>AMs tipo por um classificador de célula (ver Tabela de materiais) diretamente em meio de cultura de 1ml AM humana ou 1 mL de tampão de Lise suplementado com 10 µ l/mL de β-Mercaptoetanol.</li>
</ol>4. in vitro cultura da AMs
<ol><li>Seguindo a classificação de célula, recolher as células por centrifugação a 250 x g a 4 ° C por 10 min.</li>
	<li>Resuspenda AMs humanas em 1 mL meio de cultura humano AM [Roswell Park Memorial Institute médio (RPMI) suplementado com 10% de 1640 FBS, 20% L-929 cultura sobrenadante (como uma fonte de colônia de macrófagos (M-CSF), fator de concentração final de 100 U/mL), piruvato de sódio 1 mM, 10 mM de ácido N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic (HEPES) e 1 x penicilina/estreptomicina (opcional)]. Da mesma forma, resuspenda mouse AMs em 1ml rato meio de cultura AM [DMEM suplementado com 10% FBS, 20% L-929 cultura sobrenadante (como uma fonte de M-CSF, concentração final de 100 U/mL), 1 mM piruvato de sódio, 10 mM HEPES e 1x penicilina/estreptomicina (opcional )].</li>
	<li>Enumerar as trypan azul-excluindo células viáveis por um contador de célula e ajustar a concentração de células viáveis em 1 x 106/mL. Nota: Idealmente, acima de 5 x 105 AMs pode ser isolado de BAL fluido coletado de um rato masculino C57BL/6 de 8-10 semanas. O rendimento do AM do digest de pulmão do rato é variável e pode ser um pouco menos do que o fluido do mouse BAL. Rendimento de AM de fluido humano BAL depende do volume do fluido BAL, o volume total de soro usado na lavagem e a condição clínica do paciente.</li>
	<li>Baseado no rendimento e necessidade experimental, as sementes das células em câmara slides, pratos de cultura ou frascos de cultura. Idealmente, as sementes do AMs em 1 x 105/mL, com média de 10 mL em um frasco de cultura de tecidos de T25.</li>
	<li>Incube as células numa incubadora umidificado de 37 ° C com 5% CO2 atmosfera. Nota: AMs vai crescer como células aderentes e são de crescimento extremamente lento (tempo de duplicação > 7 dias).</li>
	<li>Em 24 horas após a semeadura, AMs devem ser aderente e não irá ser desanexada por uma mudança suave de meio de cultura. Remover o meio por aspiração de uma extremidade e reabasteça com meio fresco, pre-aquecido a 37 ° C. Nota: As células agora podem ser usadas para avaliar a fisiologia de AM ou os efeitos de estímulos. AMs cultivadas em slides câmara podem convenientemente ser manchadas com anticorpos apropriados e são ideais para visualização microscópica. Para avaliação de fluxo cytometric, AMs são colhidos pela incubação com célula não enzimáticos, dissociando a solução.</li>
	<li>Aspire o meio de cultura e adicionar dissociando solução suficiente para cobrir a superfície de cultura (aproximadamente 2 mL por frasco T25) e incubar a 37 ° C por 5-10 minutos. Não há raspagem será necessário, e não é aconselhável raspar as células mecanicamente. Bata levemente os lados, adicionar 1 a 2 mL de tampão de fluxo/classificação e suavemente Pipetar para cima e para baixo para desanexar a maioria das células aderentes.</li>
	<li>Para estudos de RNA, lyse as pilhas diretamente sobre o prato de cultura pela adição de Lise.</li>
</ol>

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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=Zbtb7a+induction+in+alveolar+macrophages+is+implicated+in+anti-HLA-mediated+lung+allograft+rejection.">Zbtb7a induction in alveolar macrophages is implicated in anti-HLA-mediated lung allograft rejection.</a> Sci Transl Med. 9 (398), (2017).</li><li>Misharin, A. V., Morales-Nebreda, L., Mutlu, G. M., Budinger, G. R., Perlman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+macrophages+and+dendritic+cell+subsets+in+the+mouse+lung.">Flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung.</a> Am J Respir Cell Mol Biol. 49 (4), 503-510 (2013).</li><li>Kopf, M., Schneider, C., Nobs, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+function+of+lung-resident+macrophages+and+dendritic+cells.">The development and function of lung-resident macrophages and dendritic cells.</a> Nat Immunol. 16 (1), 36-44 (2015).</li><li>Eguiluz-Gracia, I., 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=Long-term+persistence+of+human+donor+alveolar+macrophages+in+lung+transplant+recipients.">Long-term persistence of human donor alveolar macrophages in lung transplant recipients.</a> Thorax. 71 (11), 1006-1011 (2016).</li><li>Yu, Y. A., 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=Flow+cytometric+analysis+of+myeloid+cells+in+human+blood,+bronchoalveolar+lavage,+and+lung+tissues.">Flow cytometric analysis of myeloid cells in human blood, bronchoalveolar lavage, and lung tissues.</a> Am J Respir Cell Mol Biol. , (2015).</li></ol>

Os autores têm sem conflitos de interesse para declarar.

MGA é macrófagos de pulmão-residente de vida longa que povoam os pulmões começando no nascimento e duradouro sobre a extensão de vida inteira26. Seus papéis na fisiologia pulmonar7 e patologia12 e seu potencial para prever de auto-imunidade pulmonar24 foram reconhecidos. Porque AMs tem uma presença de longo prazo em pulmões11,27 e porque eles estão envolvidos...

O microambiente pulmonar é um ecossistema complexo exclusivamente com uma conduta de ar elaborada e vasculatura. O ar inalado viaja através da traqueia e inúmeras ramificações dos brônquios e bronquíolos antes de atingir os alvéolos, onde ocorre a troca de gás de ar-sangue. Devido à interação direta com a atmosfera, a superfície respiratória requer proteção contra os efeitos potencialmente prejudiciais de partículas suspensas no ar e poluentes. Uma série de barreiras físicas, químicas e imunológicas protege os pulmões. Nomeadamente, a implantação dos fagócitos na superfície respiratória serve um sistema importante primeira linha de defesa. Macrófagos alveolares (AMs) são um tipo de fagócitos residente no pulmão, e eles compõem a grande maioria do grupo de macrófagos pulmonares. Como seu nome sugere, AMs são principalmente localizadas para o lúmen alveolar e ocorrem como células sésseis que constantemente a atmosfera ambiente da amostra e comunicar-se com o epitélio alveolar1. No estado estacionário, pulmões, mais de 95% dos fagócitos no espaço alveolar são AMs2, cuja composição pode alterar devido a inflamação, infecção ou exposição crônica aos poluentes.
AMs participarem em uma ampla gama de funções que podem ser locais para os pulmões e/ou de importância sistêmica. Por exemplo, AMs são essenciais para o desenvolvimento e o bom funcionamento dos pulmões; vigilância imunológica; e liberação de restos celulares, invadindo a patógenos e partículas inaladas3,4,5,6,7. Alvo de depleção de AMs é conhecida por prejudicar a liberação de vírus respiratórios e bactérias4,8. Além de seu papel como fagócitos e um defensores da primeira linha da homeostase pulmonar, AMs são conhecidos por funcionar como células apresentadoras de antígeno em suscitar T célula imunidade9, potencializando a eficácia da vacina intranasal10 e influenciando a auto-imunidade restrição pulmonar após transplante de pulmão11,12. Deficiência na função AM tem sido associada a lipoidoproteinose alveolar pulmonar (PAP), uma condição resultante de uma mutação genética, malignidade ou infecção que prejudica o apuramento de surfactantes pulmonares13,14. Transplante de AMs agora está sendo explorada como uma abordagem terapêutica para o tratamento de PAP 15,16.
MGA é conhecidas que se originam durante a embriogênese e a persistir nos pulmões durante toda a vida sem ser substituído por leucócitos2,17de circulação. Embora, volume de negócios AM é indetectável nos pulmões homeostáticos, diferentes níveis de volume de negócios AM têm sido relatados em determinadas condições clínicas, incluindo a infecção pela gripe vírus4, mieloablativo irradiação18, exposição a endotoxina 19e20anos. MGA é acreditadas para auto renovar através de um baixo grau de proliferação de17,21, mas alguns estudos recentes afirmam que os monócitos podem dar origem a uma população de pulmão intravascular macrófagos22,23 , sob condições experimentais, mas a funcionalidade destes recém-convertidos macrófagos pulmonares ainda têm de ser definidos em doenças pulmonares. Além disso, compreender o limiar de estímulo no contexto de ativação AM é uma área potencialmente interessante, como o pulmão tenta preservar um equilíbrio entre os sinais inflamatórios e a maquinaria imunorreguladores.
As alterações fisiológicas ou patológicas que levam à perda do Regulamento imune são importantes para avaliar em vários ambientes clínicos (por exemplo, infecções respiratórias, doença inflamatória pulmonar e doenças pulmonares fibróticas). Não obstante, AMs são cada vez mais reconhecidos como indicadores ou mesmo determinantes da saúde pulmonar11,24. Atualmente, existem protocolos não unificados disponível para colheita, caracterizando, e/ou manutenção AMs com os humanos e modelos pré-clínicos de murino. Falta de um consenso sobre AM precursores e fenótipos e ausência de uma metodologia detalhada tinham sido grande bloqueio em decifrar função (ões) de AM na doença e na saúde pulmonar. O seguinte protocolo oferece uma identificação definitiva, isolamento e em vitro cultura estratégia extremamente avançar a compreensão do comportamento de AM e facilitar estudos de diagnósticos e terapêuticos AM-alvo.

<table border="0" cellpadding="0" cellspacing="0" width="910">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Non-enzymatic cell dissociating solution</td>
			<td style="width:223px;">Millipore-Sigma</td>
			<td style="width:220px;">C5789</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puralube Vet Ointment</td>
			<td style="width:223px;">Dechra</td>
			<td>620300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">22G Catheter&#160;</td>
			<td style="width:223px;">Terumo Medical Products</td>
			<td>SR-OX2225CA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-0 Non-absorbable silk braided suture&#160;</td>
			<td style="width:223px;">Kent Scientific</td>
			<td>SUT-15-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s phosphate buffered saline&#160;</td>
			<td style="width:223px;">Corning</td>
			<td style="width:220px;">21-031-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Fc block&#160;</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysis buffer (PureLink RNA Kit)</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>12183018A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">b-Mercaptoethanol&#160;</td>
			<td style="width:223px;">Millipore-Sigma</td>
			<td>M6250&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACSAria II cell sorter&#160;</td>
			<td style="width:223px;">BD Biosciences</td>
			<td style="width:220px;">644832</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine&#160; (Ketathesia)</td>
			<td style="width:223px;">Henry Schein</td>
			<td style="width:220px;">56344</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine&#160; (AnaSed)</td>
			<td style="width:223px;">Akorn</td>
			<td style="width:220px;">139-236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td style="width:223px;">Corning</td>
			<td style="width:220px;">10-040-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:223px;">Corning</td>
			<td style="width:220px;">10-017-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liberase TL&#160;</td>
			<td style="width:223px;">Millipore-Sigma</td>
			<td style="width:220px;">5401020001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I</td>
			<td style="width:223px;">Millipore-Sigma</td>
			<td>AMPD1-1KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100&#956;m cell strainer&#160;</td>
			<td style="width:223px;">Corning</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Human Fc block</td>
			<td>BD Biosciences</td>
			<td>564220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">EDTA</td>
			<td style="width:223px;">Corning</td>
			<td>46-034-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Countess II Automated Cell Counter</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Trypan Blue Solution</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">HEPES</td>
			<td style="width:223px;">Corning</td>
			<td>25-060-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">L-929 cell line</td>
			<td>American Type Culture Collection</td>
			<td>ATCC,&#160;CCL-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin&#160;</td>
			<td style="width:223px;">Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Pyruvate</td>
			<td style="width:223px;">Corning</td>
			<td>25-000-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T25 Tissue culture flask</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>156367</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 mm culture dish&#160;</td>
			<td style="width:223px;">Millipore-Sigma</td>
			<td>CLS3261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">15 mL Conical tube&#160;</td>
			<td style="width:223px;">Corning</td>
			<td style="width:220px;">352097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">50 mL Conical tube&#160;</td>
			<td style="width:223px;">Corning</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSRFortessa cell analyzer</td>
			<td>BD Biosciences</td>
			<td>657669</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">FlowJo</td>
			<td style="width:223px;">FlowJo</td>
			<td>v10.4</td>
			<td>Analysis Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD45 (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>147709</td>
			<td>Clone I3/2.3, FITC conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD11b (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>101228</td>
			<td>Clone M1/70, PerCP/Cy5.5 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD11c (Mouse)</td>
			<td>BD Biosciences</td>
			<td>565452</td>
			<td>Clone N418, BV 421 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-I-Ab (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>116420</td>
			<td>Clone AF6-120.1, PE/Cy7 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-Siglec-F (Mouse)</td>
			<td style="width:223px;">BD Biosciences</td>
			<td>562757</td>
			<td>Clone E50-2440, PE-CF594 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-Siglec-H (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>129605</td>
			<td>Clone 551, PE conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-F4/80 (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>123118</td>
			<td>Clone BM8, APC/Cy7 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-Ly-6C (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>128035</td>
			<td>Clone HK1.4, BV605 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD64 (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>139311</td>
			<td>Clone X54-5/7.1, BV711 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD24 (Mouse)</td>
			<td style="width:223px;">BD Biosciences</td>
			<td>563115</td>
			<td>Clone M1/69, BV510 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD103 (Mouse)</td>
			<td style="width:223px;">BD Biosciences</td>
			<td>745305</td>
			<td>Clone OX-62, BV650 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD317 (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>127015</td>
			<td>Clone 927, APC conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CXCR1 (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>149029</td>
			<td>Clone SA011F11, BV785 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD45 (Human)</td>
			<td style="width:223px;">Biolegend</td>
			<td>304017</td>
			<td>Clone HI30, AF488 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD11b (Human)</td>
			<td style="width:223px;">Biolegend</td>
			<td>101216</td>
			<td>Clone M1/70, PE/Cy7 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-HLA-DR (Human)</td>
			<td style="width:223px;">Biolegend</td>
			<td>307618</td>
			<td>Clone L243, APC/Cy7 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD169 (Human)</td>
			<td style="width:223px;">Biolegend</td>
			<td>346008</td>
			<td>Clone 7-239, APC conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD206 (Human)</td>
			<td style="width:223px;">Biolegend</td>
			<td>321106</td>
			<td>Clone 15-2, PE conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD163 (Human)</td>
			<td style="width:223px;">Biolegend</td>
			<td>333612</td>
			<td>Clone GHI/61, BV421 conjugated</td>
		</tr>
	</tbody>
</table>

isolation and in vitro culture of murine and human alveolar macrophages

Os macrófagos alveolares são macrófagos terminalmente diferenciados, pulmão-residente de origem pré-natal. Os macrófagos alveolares são únicos em sua longa vida e o seu importante papel no desenvolvimento pulmonar e função, bem como suas respostas pulmão-localizada a infecção e inflamação. Até à data, nenhum método unificado para identificação, isolamento e manipulação dos macrófagos alveolares com os humanos e ratos existe. Esse método é necessário para estudos sobre essas células imunes inatas importantes em várias configurações experimentais. O método descrito aqui, que pode ser facilmente adoptado por qualquer laboratório, é uma abordagem simplificada para colheita de macrófagos alveolares de líquido de lavagem broncoalveolar ou de tecido pulmonar e mantê-los em vitro. Porque os macrófagos alveolares ocorrem principalmente como células aderentes no alvéolo, o foco desse método é desalojá-los antes da colheita e identificação. O pulmão é um órgão altamente vascularizado, e vários tipos de células de origem mieloide e linfoide habitam, interagem e são influenciados pelo microambiente do pulmão. Usando o conjunto de marcadores de superfície descrita aqui, pesquisadores podem facilmente e de forma inequívoca distinguir outros leucócitos macrófagos alveolares e purificá-los para aplicações a jusante. O método de cultura desenvolvido neste documento suporta ambos humanos e macrófagos alveolares para multiplicação in vitro de rato e é compatível com estudos celulares e moleculares.

A abordagem de fluxo cytometric identificar rato AMs é mostrada na Figura 1. Isso inclui a análise de um conjunto mínimo de marcadores de superfície necessárias na distinção entre AMs de outros fagócitos residentes pulmonar ou pulmão infiltrando. É necessária análise diferencial de identificar positivamente AMs de intersticiais macrófagos, células dendríticas, neutrófilos, monócitos e macrófagos derivados de monócitos pulmonares que ocorrem...

Watch this Scientific Journal Video about Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages at JoVE.com

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolamento e cultura In Vitro de macrófagos alveolares murino e humanos

Esta comunicação descreve metodologias para isolamento e cultura de macrófagos alveolares dos humanos e modelos murino para fins experimentais.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

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JoVE Journal

Immunology and Infection

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

23.4K visualizações.  St. Joseph's Hospital and Medical Center. Esta comunicação descreve metodologias para isolamento e cultura de macrófagos alveolares dos humanos e modelos murino para fins experimentais.

Vídeo: Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Isolamento T de linfócitos humanos, Cultura e Análise da Migração In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Imunologia e Infecção

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

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

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

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

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

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Estabelecimento de um<em&gt; In vitro</em&gt; Sistema para estudar o comportamento intracelular de<em&gt; Candida glabrata</em&gt; Em humanos THP-1 macrófagos

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Isolamento de monócitos humanos por duas vezes centrifugação em gradiente e sua diferenciação de macrófagos em revestidos de teflon Cultura de Células Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

Avaliar a atividade anti-fúngica isolada de macrófagos alveolares por microscopia confocal

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Isolamento de Murino macrófagos peritoniais de Cumprir a análise de expressão gênica Após Toll-like receptors Estimulação

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

Cultura de macrófagos macrófagos Colony estimulante fator diferenciado Humano monócitos derivados

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Quantificar o humano Monocyte Chemotaxis In Vitro e linfócitos murino tráfico Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolamento de Salmonella typhimurium-contendo Phagosomes de macrófagos

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Isolation, Transfection, and Culture of Primary Human Monocytes

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4+ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14+ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.

Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.

Isolamento, Transfecção e Cultura dos Monócitos Humanos Primários

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. ...

Isolamento e cultura In Vitro de macrófagos alveolares murino e humanos

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