Este trabalho foi financiado pelo piloto e concessão Estudo de Viabilidade da UCLA-CURE Center, a Fundação de Crohn e Colite da América Prêmio de Desenvolvimento de Carreira (# 2691) e do Instituto Nacional de Saúde NIDDK K01 (DK084256) financiamento para Hon Wai Koon. Óssea operação irradiação medula foi assistido por Bernard Levin e Cozinha Scott, da UCLA Center for AIDS Research Rato / instalação humanos fundamentais Quimera. Operação de citometria de fluxo foi assistida pela UCLA instalação Núcleo do vetor.

1. Antes-de-começar Considerações Técnicas <ol><li> Recomendamos o uso de ambos os C57/BL6 camundongos selvagens e camundongos knockout para o experimento porque os ratos com moléculas de CD correspondentes pode ser comprado de fornecedores do mouse principais. </li><li> Uma vez que é difícil controlar os ratinhos do tipo selvagem e ratinhos knockout derivadas de células dadoras após transplante de medula óssea, é necessária a utilização de ratinhos CD45.1 para representar ratinhos de tipo selvagem. (CD45.1 C57/SJL WT ratos Jackson estoque Laboratories # 002014). </li><li> A maioria das colônias do rato são CD45.2. Camundongos knockout nossas pertencem a CD45.2 genótipo (alternativamente uma fonte CD45.2 ratinhos WT: Jackson Laboratories stock # 000664). A determinação do CD45.1 ou CD45.2 genótipo pode ser realizada por citometria de fluxo de medula óssea e sangue periférico, como mencionado na seção 7 deste protocolo. </li><li> Camundongos após irradiação ter comprometido a imunidade. Mantenha ratos em instalação livre de patógenos. </li><li> Operação de irradiador requer especial de segurança clearance e treinamento. Para economizar tempo nos processos de formação e aprovação, o melhor é colaborar com um laboratório com técnico qualificado que é capaz de operar o irradiador. </li><li> Muitas instituições têm laboratórios de citometria de fluxo com técnicos experientes. Para minimizar problemas com a operação de citometria de fluxo, é melhor discutir o seu fluxo de citometria de plano experimental com o técnico experiente antes de começar. </li><li> Os ratos foram atribuídos desta maneira (10 ratos por grupo): </li></ol><table border="1"><tbody><tr><td> Grupo </td><td></td><td> Doador de medula óssea para o destinatário </td><td> Tratamento </td></tr><tr><td> A </td><td> BM troca </td><td> CD45.1 WT para CD45.2 KO </td><td> DSS colite </td></tr><tr><td> B </td><td></td><td></td><td> Controle normal de água </td></tr><tr><td> C </td><td> BM troca </td><td> CD45.2 KO para CD45.1 WT </td><td> DSS colite </td></tr><tr><td> D </td><td></td><td></td><td> Controle normal de água </td></tr><tr><td> E </td><td> Impostura </td><td> CD45.1 WT para CD45.1 WT </td><td> DSS colite </td></tr><tr><td> F </td><td></td><td></td><td> Controle normal de água </td></tr><tr><td> G </td><td> Impostura </td><td> CD45.2 KO para CD45.2 KO </td><td> DSS colite </td></tr><tr><td> H </td><td></td><td></td><td> Controle normal de água </td></tr></tbody></table> * Uma breve ilustração do protocolo experimental é apresentado na Figura 1. 2. Irradiação Mice destinatário <ol><li> Ratos são criados e mantidos em instalações livres de agentes patogénicos. Coloque em ratos torta irradiação autoclavado com filtro. Coloque um rato em cada slot de torta de irradiação. Coloque a torta para o irradiador e certifique-se o prato e torta estão girando. </li><li> Ligue a bomba de arpara ventilação. Feche a porta do irradiador e bloqueá-lo. </li><li> Irradiar os murganhos com 1000 rad (o qual é equivalente a 10 Gy) em torno de 10 minutos (dependendo da fonte de radiação e de semi-vida). A partir deste ponto de tempo, os ratinhos irradiados são imuno-comprometidos e têm fraca imunidade contra a infecção. Após a irradiação, leve a torta para fora e colocar em um recipiente estéril para o transporte de biossegurança gabinete em instalações para animais. Evitar expor os ratos ao ambiente externo para minimizar a possibilidade de infecção. </li><li> Em biossegurança gabinete, transferir os ratos em gaiolas de rato autoclavados com cama autoclavado (4 ratos por gaiola Adicionar água estéril novo com suspensão sulfatrim -. 3,12 ml por 100 ml de água). </li><li> Enrole a garrafa de água com uma folha de alumínio como os antibióticos são sensíveis a luz. Agite para misturar o frasco da solução sulfatrim bem antes de carregá-lo para a jaula. </li></ol> 3. Extracção doador de medula óssea <ol><li> Sacrificar os ratos com dióxido de carbono ou isoflurdade e, em seguida, mergulhe em 1:200 solução desinfetante. Pulverizar os ratos com etanol a 70%, para molhar a pele e dissecar ossos abertos. </li><li> Mergulhe os instrumentos de dissecção em etanol de 70% para a esterilização. </li><li> Realizar a dissecação em um armário de biossegurança. Use os instrumentos de dissecção estéreis para expor o úmero, fêmur, tíbia e fíbula ossos, livre de fixar os músculos e ligamentos. </li><li> Cortar abrir ambas as extremidades dos ossos para mostrar a medula óssea vermelha. Manter os ossos com uma pinça. Lave a medula óssea vermelha com PBS frio, com seringa de 3 ml e agulhas 25G para uma placa de Petri. Lave a partir de ambas as extremidades dos ossos, para se obter as células da medula óssea mais. </li><li> Use 6 seringas ml e 18G1 / 2 agulhas para quebrar plugues de medula óssea vermelha por aspiração repetida e ejeção. Transferência de medula óssea com tubos de 50 ml Falcon. </li><li> Girar a medula óssea para baixo em 1800 rpm durante 5 min a 4 ° C. Elimine o sobrenadante, ressuspender pellet em vortex por 5-10 segundos. </li><li> Diluir 10X de tampão de lise de RBC com água estéril. Anúnciod 1X tampão de lise (5 ml por rato doador), vortex novamente durante 5 segundos e manter exactamente 3 minutos à temperatura ambiente. </li><li> Após 3 min, encher os tubos com 20 ml de PBS frio para parar a lise e mistura-se. Despeje o conteúdo através de um filtro de células 40 mM diretamente em um novo tubo de 50 ml Falcon. </li><li> Enxaguar os tubos originais com 5 ml de PBS e transferir para o coador de células também. Ateste para 50 ml com PBS e agitar. </li></ol> 4. Contagem de células de doadores de medula óssea <ol><li> Adicionar 100 ul de azul de tripano a um tubo Eppendorf a 100 ul de suspensão de medula óssea, para um tubo de Eppendorf e misturar. Pipetar a mistura de células coradas com um hemocitómetro. </li><li> A quantidade de células é calculado através da contagem de células totais vivo de medula óssea em tubos Falcon = número de células não coradas em 16 quadrados x 2 (devido a diluição azul de Trypan) x 10,000 x 50 ml </li><li> Girar as células da medula óssea, em tubos de 50 ml Falcon a 2.000 rpm durante 5 min a 4 ° C. </li><li> Remover o sobrenadante. Baseadosobre as contagens de células totais, ressuspender o pellet com PBS a 1 x 10 células por ml 8 </li></ol> 5. Infusão de ratinhos receptores <ol><li> Aqueça os ratos receptores sobre uma almofada de calor e sob uma lâmpada de aquecimento. Coloque os ratos beneficiários no limitador. Injectar 1 x 10 7 células por rato em 100-200 ul por via intravenosa. </li><li> Injecção de medula óssea do dador deve ser feita entre 4-24 horas após a irradiação. </li><li> Mantenha-se quatro camundongos injetados por gaiola. Manter os camundongos injetados destinatário em gaiolas autoclavados com água-tratamento por antibióticos nas primeiras 4 semanas em biotério patógeno livre e deixar os ratos recuperar a imunidade. Gaiolas de mudança, de água tratado com antibióticos e alimentos a cada 4 dias para manter a higiene. </li></ol> 6. Indução de colite e Avaliação de Colite <ol><li> 4 semanas após a irradiação e transplante de medula óssea, mude para a água regular sem antibióticos e manter os ratos para mais 2 semanas para recuperar o seu normalgut microflora. </li><li> 6 semanas após a injeção de medula óssea, medir o peso corporal inicial. Alguns ratos recebem 5% de sulfato de dextrano em beber água ad libitum para induzir a colite. Alguns ratos são dadas água potável regular apenas como grupos de controle normais. </li><li> 5 dias depois, retirar o sangue periférico, a transferência para tubos revestidos de heparina (Vacutainer) e manter-se em gelo. A piscina do sangue de 4 ratos para um tubo. Combinar 300 ul de sangue com 300 ul de tampão de coloração de células = pi 600. Dividir o sangue para tubos Eppendorf 5, cada um com 100 ul. </li><li> Sacrificar os ratos com dióxido de carbono ou isoflurano. Avaliar os escores de danos macroscópicos (consultar a outra publicação vídeo JOVE para detalhes 1). Medir variação de peso. Medida comprimento do intestino e espessura com paquímetro digital. Observe textura das fezes (duro, macio ou com sangue). Determine de sangue oculto nas fezes com Hemooccult. Dissecar tecidos do cólon e fixar em formol para a H &amp; E coloração. </li><li> Dissecar um fêmur bone por rato, extrair medula óssea e lave a medula óssea com frio PBS através de agulha 25G e uma seringa ml. Piscina de medula óssea 4 fichas de um tubo e manter em gelo. </li><li> Despeje a medula óssea, para uma placa de Petri, a ficha de cisalhamento de medula óssea com uma agulha de 18G e 5 vezes ml seringa até vários nenhuma ficha medula óssea está presente. </li><li> Girar com 1.500 rpm durante 5 min a 4 ° C. Remover o sobrenadante e ressuspender com 600 ul de tampão de coloração de células. Dividir a medula óssea ressuspenso em 5 tubos de Eppendorf, cada um com 100 ul. </li></ol> 7. Inspeção de Qualidade de Transplante de Medula Óssea por citometria de fluxo <ol><li> Rotular cada tubos de amostras de sangue ou de medula óssea # 1-5: </li><li> Preparar a mistura de anticorpo para cada respectivo tubo no escuro. </li></ol> # 1 Não anticorpo # 2 30 ul de controlo de isotipo FITC + 30 uL de controlo de isotipo PE + 15 ul de bloqueio CD16/32 # 3 30 ul FITC CD45.1 Ab + 15 ul de bloqueio CD16/32 # 4 30 PE ulCD45.2 Ab + 15 ul de bloqueio CD16/32 # 5 30 ul de Ab FITC CD45.1 + 30 uL PE CD45.2 Ab + 15 ul de bloqueio CD16/32 <table border="1"><tbody><tr><td> Adicione nada a amostra de sangue ou de medula óssea # 1 </td></tr><tr><td> Adicionar 5 uL de mistura de anticorpo # 2 para cada amostra de sangue ou de medula óssea # 2 </td></tr><tr><td> Adiciona-se 3 uL de mistura de anticorpo # 3 para cada amostra de sangue ou de medula óssea # 3 </td></tr><tr><td> Adiciona-se 3 uL de mistura de anticorpo # 4 para cada amostra de sangue ou de medula óssea # 4 </td></tr><tr><td> Adicionar 5 uL de mistura de anticorpo # 5 para cada amostra de sangue ou de medula óssea # 5 </td></tr></tbody></table><ol start="3"><li> Manter no escuro em gelo durante 30 min. </li><li> Adicionar 2 ml de 1X tampão de lise de glóbulos vermelhos para cada tubo. Manter no escuro em gelo durante 15 min. </li><li> Girar as células a 1500 rpm, 5 min a 4 ° C. (GH rotor 3.8, 1.500 rpm = 350 xg) Remover o sobrenadante. Ressuspender o sedimento com 500 ul de tampão de coloração de células no brevemente, em seguida escuro vortex. </li&gt; <li> Verifique a CD45.1 (representando WT) e CD45.2 (representando KO) no sangue imunologicamente e amostras de medula óssea por citometria de fluxo. Selecione FITC para representar WT CD45.1 e PE para representar KO CD45.2. </li><li> Analisar os resultados de citometria de fluxo por software FlowJo. Calcular a proporção de FITC: PE ou PE proporção: relação FITC para determinar se o genótipo doador é genótipo dominante no sangue e na medula óssea. </li></ol>

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Não há conflitos de interesse declarados.

Este transplante de medula óssea abordagem é adequada para a pesquisa de imunologia colite, infecção, cancro, obesidade e outras doenças. Este transplante de medula óssea experimento é necessária quando o gene de interesse é expresso tanto na medula óssea e células derivadas de medula óssea não derivados e o gene de interesse, se suspeita de mediar doença por células a partir de qualquer população. Por exemplo, o péptido antimicrobiano cathelicidin é mostrado para modular a colite aguda. Mas este é e...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Tampão de coloração de células </td><td> Biolegend </td><td> # 420201 </td></tr><tr><td> 10X tampão de lise de RBC </td><td> Biolegend </td><td> # 420301 </td></tr><tr><td> FITC rato isotipo controle </td><td> Biolegend </td><td> # 400207 </td></tr><tr><td> PE rato isotipo controle </td><td> Biolegend </td><td> # 400211 </td></tr><tr><td> PE anti-rato CD45.2 </td><td> Biolegend </td><td> # 109807 </td></tr><tr><td> FITC anti-rato CD45.1 </td><td> Biolegend </td><td> # 110705 </td></tr><tr><td> Anti-rato CD16/32 bloqueio </td><td> Biolegend </td><td> # 101301 </td></tr><tr><td> 40 filtro de células mM </td><td> Fisherbrand </td><td> # 22363547 </td></tr><tr><td> PBS 1X </td><td&gt; MP Biomedicals </td><td> # 1860454 </td></tr><tr><td> Heparina </td><td> Fisherbrand </td><td> # BP2425 </td></tr><tr><td> Sulfatrim (SMZ) </td><td> Qualitest </td><td> NC9242720 (Fisher) </td></tr><tr><td> Lysol IC </td><td> Andwin Científico </td><td> NC9745686 (Fisher) </td></tr><tr><td> Vaccutainer </td><td> BD </td><td> # 8000813 </td></tr><tr><td> Rato limitador </td><td> Braintree Científica </td><td> # TV-150 </td></tr><tr><td> Irradiador </td><td> JL Pastor e Associados </td><td> Mark I 68A </td></tr><tr><td> Citómetro </td><td> BD </td><td> BD FACSCanto II </td></tr><tr><td> Tubo de ensaio de fluxo citómetro </td><td> Falcão </td><td> # 352052 </td></tr><tr><td> Paquímetro digital </td><td> Fisherbrand </td><td> 14-648-17 </td></tr><tr><td> Hemoccult TIC </td><td> Beckman Coulter </td><td> G0328QW </td></tr&gt; </tbody></table>

differentiating functional roles of gene expression from immune and non-immune cells in mouse colitis by bone marrow transplantation

Para compreender o papel de um gene no desenvolvimento de colite, foram comparadas as respostas dos ratinhos de tipo selvagem e gene-de-interesse camundongos knockout para deficientes colite. Se o gene de interesse é expresso em ambas as células de medula óssea e células derivadas de medula óssea não derivadas do hospedeiro, no entanto, é possível distinguir a função de um gene de interesse em células de medula óssea e não derivadas de medula óssea células derivadas pela técnica de transplante de medula óssea. Para alterar o genótipo de medula óssea derivada de células de ratos, a medula óssea original de ratinhos receptores foram destruídas por irradiação e, em seguida, substituído por medula óssea de dador novo genótipo diferente. Quando ratinhos do tipo selvagem da medula óssea do dador foi transplantado para ratos knockout, podemos gerar ratinhos knockout de tipo selvagem, com a expressão de genes em células da medula óssea derivadas. Alternativamente, quando ratinhos knockout de medula óssea do dador foi transplantado para ratos do tipo selvagem destinatário, ratinhos de tipo selvagem, sem gene de interesse que expressama partir de células de medula óssea derivadas foram produzidos. No entanto, o transplante de medula óssea pode não ser 100% completa. Portanto, utilizou-se aglomerado de diferenciação (CD) moléculas (CD45.1 e CD45.2) como marcadores de células do doador e do receptor para controlar a proporção de medula óssea do dador de células derivadas de ratinhos receptores e do sucesso do transplante de medula óssea. Ratinhos de tipo selvagem com CD45.1 ratos knockout com genótipo e CD45.2 genótipo foram utilizados. Após a irradiação dos ratos receptores, as células de medula óssea de dadores de diferentes genótipos foram infundidos nos ratinhos receptores. Quando a medula óssea novo regenerado para assumir a sua imunidade, os animais foram desafiados por agente químico (dextran sulfato de sódio, 5% de DSS) para induzir a colite. Aqui também mostraram que o método para induzir a colite em ratos e avaliar o papel do gene de interesse expresso a partir de células da medula óssea derivadas. Se o gene de interesse, a partir de células de osso derivadas desempenha um papel importante no desenvolvimento da doença (tal como E. colitis), o fenótipo dos ratinhos receptores de transplante de medula óssea pode ser significativamente alterada. No final das experiências de colite, a medula óssea derivadas de células no sangue e na medula óssea foram marcadas com anticorpos contra CD45.1 e CD45.2 e a sua razão quantitativa de existência pode ser utilizada para avaliar o sucesso do transplante de medula óssea por citometria de fluxo. O transplante de medula óssea de sucesso deve mostrar uma grande maioria dos genótipo doador (no termo do marcador CD molécula) ao longo de genótipo receptor, tanto na medula óssea e no sangue de ratos receptores.

Se o gene de interesse, desempenha um papel importante em células imunitárias durante o desenvolvimento de colite, os murganhos que receberam medula óssea de genótipo diferente através de transplante de medula óssea (WT KO ou KO para a WT) deve ter uma resposta alterada a colite DSS. Um dos parâmetros mais importantes para determinar a gravidade da colite é a coloração de H &amp; E de tecidos do cólon. Alterações no tecido do cólon estrutura e sinais de inflamação pode ser avaliado quantitativamente pela...

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

O transplante de medula óssea oferece uma maneira de alterar o genótipo de células derivadas de medula óssea. Se o gene de interesse é expresso em ambas as células de medula óssea e células derivadas de medula óssea não derivados, transplante de medula óssea pode mudar as células de medula óssea derivadas de um genótipo diferente sem alterar a non-bone marrow genótipo celular derivada.

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 ...

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Research

JoVE Journal

Immunology and Infection

17.4K visualizações.  The University of California Los Angeles, Los Angeles. O transplante de medula óssea oferece uma maneira de alterar o genótipo de células derivadas de medula óssea. Se o gene de interesse é expresso em ambas as células de medula óssea e células derivadas de medula óssea não derivados, transplante de medula óssea pode mudar as células de medula óssea derivadas de um genótipo diferente sem alterar a non-bone marrow genótipo celular derivada.

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

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

Isolation of Functional Cardiac Immune Cells

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

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

Isolamento de células do sistema imunológico funcional cardíaca

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

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Isolamento, purificação e Rotulagem de neutrófilos da medula óssea do rato de Estudos funcionais e Experimentos de transferência adotiva

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Acompanhando Rato Medula Óssea monócitos<em&gt; In Vivo</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Geração e identificação de GM-CSF macrófagos derivados e dendríticas células alveolares do tipo de ratinho de Medula Óssea

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Classificação de células activadas por fluorescência Para a purificação de células dendríticas plasmocitï¿½des do rato de Medula Óssea

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Identificação e isolamento de oligopotentes e confirmada por linhagem mieloides progenitores da medula óssea de rato

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

Isolamento de células mononucleares de lamina propria de cólon murino usando Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

Plataforma de transplante de medula óssea para investigar o papel das células dendríticas na doença enxerto-versus-hospedeiro

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

Procedimentos de transplante de medula óssea em camundongos para estudar hematopoiese clonal

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...