Os autores agradecem a Anne Daron e Pierre Louis Loyher de assistência editorial, o Plateforme Imagerie Pitié-Salpêtrière (PICPS) para a assistência com o microscópio de dois fótons e do Mecanismo de animal &quot;NAC&quot; e Camille Baudesson para ratos reprodutores assistência. A investigação conducente a estes resultados foi financiada pelo Sétimo Programa-Quadro da Comunidade Europeia (FP7 / 2007-2013) ao abrigo do contrato de concessão n ° 304810 - RAIDs, e n ° 241440-Endostem, do Inserm, pela Université Pierre et Marie Curie &quot;Emergence &quot;, de la&quot; Ligue contre le câncer &quot;, de&quot; Association pour la Recherche sur le Cancer &quot;e de&quot; Agence Nationale de la Recherche &quot;Emergence Programa 2012 (ANR-EMMA-050). PH foi apoiada por la &quot;Ligue contre le cancer&quot;.

NOTA: Todos os protocolos experimentais foram aprovados pelo Experimentação Animal e Ética Comitê de francês e validado pelo &quot;Serviço de Proteção ao et Santé Animales, Environnement&quot; com o número de A-75-2065. Os tamanhos das amostras são escolhidos para assegurar a reprodutibilidade dos ensaios, e de acordo com o 3R de regulação ética dos animais. 1. Preparação do Rato <ol><li> Anestesia <ol><li> Para curto período de imagem (menos do que 1 hora), anestesiar o rato com uma injecção intraperitoneal de 200 ul de uma solução salina contendo cetamina (100 mg / kg) e xilazina (10 mg / kg). </li><li> Em alternativa, por um período maior de imagiologia (até 4 horas), anestesiar o rato com uma inalação de isoflurano 2,5% vaporizado numa mistura de O 2 / N 2 O, 70/30 através de uma máscara adaptada. </li><li> NOTA: Este modo fornece para inconsciência mais estável e evita a injeção intraperitoneal de série de drogas. </li&gt; <li> Uma vez que o mouse é anestesiado, verifique se há perda de consciência por meio da estimulação da pata traseira com pinças. </li></ol></li><li> Coloração Vascular / coloração adicional <ol><li> Para a coloração vascular: injectar por via intravenosa na veia da cauda, ​​a 200 ul de rodamina-dextrano (MDA 2, 10 mg / ml em tampão de solução salina). </li><li> Para a coloração dos neutrófilos: injectar 2 ug de Ly6G-PE (clone 1A8) em 100 ul de solução salina por via endovenosa na veia da cauda. </li></ol></li><li> Imobilização <ol><li> Para a imobilização, use um suporte stereotactic custom made adaptada ao microscópio e à inhalator gás anestésico. NOTA: O titular sterotactic é um suporte de metal com dois suportes de placa de metal, sendo uma fixa e outra removível e adaptável, para apertar a cabeça do animal. </li><li> Instale a cabeça do rato entre as placas de retenção para garantir o isolamento dos movimentos respiratórios. Aperte os ouvidos entre as placas de retenção e de cabeça para esticar a sk. Verifique para a estabilidade eo bem-estar do animal respirando. </li></ol></li><li> Remover o couro cabeludo <ol><li> Usar cuidadosamente etanol a 70% para molhar o cabelo do couro cabeludo com um aplicador estéril, evitando o contacto com os olhos. </li><li> Corte a pele com tesouras e pinças estéreis para remover totalmente o couro cabeludo do trás do osso parietal até o osso frontal. Mantenha-se afastado até 3 mm de olhos e ouvidos para prevenir hemorragia extensa. Remover o tecido conjuntivo frouxo que cobre o crânio (periósteo). Lave o cabelo restante com toalha de papel embebido PBS-quente. </li></ol></li><li> O sistema de imersão configurado <ol><li> Cole um anel de borracha de 18 milímetros de diâmetro com cola cirúrgica diretamente no crânio com uma pequena agulha de calibre para controlar a distribuição. Cola de toda a periferia do anel de borracha para evitar fugas (30 mL deve ser suficiente). </li><li> Limpe o crânio debaixo do ringue pela última vez com toalha de papel PBS-encharcado e em seguida, adicione 37 ° C PBS para imersão completa docrânio. Adicionar uma gota de solução de NaCl a 0,9% ou uma pomada oftálmica específica em cada olho do rato para evitar a secura ocular. Arraste o titular estereotáxica no âmbito do objectivo. </li><li> Cerca de posicionar o campo usando o microscópio ocular através da transmissão direta. </li></ol></li></ol> 2. Dois fótons Imaging Acquisition <ol><li> Use um microscópio multiphoton juntamente com um laser de Ti: cristal de safira, que fornece 140fs pulsos de luz NIR, ajustáveis ​​seletivamente de 680 a 1080 nm, e um modulador acústico-ótico para o controle de potência do laser. Usar uma configuração incluindo 3 detectores externos não descanned (NDD) (que permitem a gravação simultânea de três canais fluorescentes) com uma combinação de dois espelhos dicróicos (565 nm e 690 nm), 565/610 e 500/550 filtros passa-banda, e um 485 filtro de passagem curta, com um plano de Apochromat × 20 (NA = 1) objetiva de imersão em água. </li><li> Defina a câmara de aquecimento para permitir uma boa homeothermy dos Anaesrato thetized., NOTA: Temperatura pode ser definido de 1 hora antes de imagem sessão para melhor estabilidade. No nosso caso, de 32 ° C foi escolhida para obter a temperatura ideal do corpo do rato anestesiado (36-37 ° C) e é mantida até 4 horas. Esta temperatura pode ser adaptado para o sistema utilizado (sonda digital pode ser utilizado para verificar este ponto). Além disso, a câmara de aquecimento é utilizada escuro / preto e cobre uma parte do sistema (placa de objectivos e motorizado) para evitar a contaminação de luz externa. </li><li> Ligue o sistema <ol><li> Ligue o laser de Ti: Safira, então todo o sistema de microscópio. Inicie o software de aquisição. Defina o intervalo de comprimento de onda do laser de 870 nm. Selecione NDD apropriado para a gravação. NOTA: No nosso caso, geração de segundo harmônico (SHG) 19 e proteína fluorescente ciano (ECFP) são detectados pelo NDD após o filtro de passagem curta 485. ECFP também é detectado pelo NDD depois do filtro passa-banda e 500/550 rodamina-dextrano é detectado por tele NDD após o filtro passa-banda 565/610. </li></ol></li><li> Ajustar as definições de aquisição <ol><li> Use o comando de imagem &quot;aquisição ao vivo&quot; do software e utilizar o controle direcional microscópio, ajustar o foco em uma região com sinal ECFP e Rhodamine. Definir a potência do laser para o mínimo para se obter a melhor relação sinal-para-ruído com um mínimo de foto-lesão. NOTA: O ajuste de potência utilizada varia de acordo com a profundidade da região de interesse. Normalmente as configurações de OMA estão situados em torno de 20%, o que representa uma potência do laser para além do objetivo em torno de 15 mW. É melhor aumentar o ganho de poder nas PMT para cada NDD para reduzir foto-branqueamento e foto-dano. Configurações de aquisição típicos são a digitalização única, com um tempo de permanência de pixel de 1,58 uS, e uma resolução de 512 x 512 pixels com uma ampliação de 1,5. </li></ol></li><li> Selecione as regiões de interesse <ol><li> Uma vez que os parâmetros de gravação são definidos, use novamente o comando de aquisição &quot;ao vivo&quot;,e examinar o tecido para escolher um (x y z,) campo desejado para ser monitorado em tempo real. NOTA: Normalmente, nós escolhemos um campo incluindo tanto vascular e áreas de parênquima. </li><li> Registre-se o campo com o comando de software &quot;posição&quot;. NOTA: Este comando memoriza x, y, z coordenadas de diferentes áreas distantes. </li><li> Selecione e registrar campos adicionais de interesse (até 3), utilizando o mesmo procedimento. </li><li> Definir um z-pilha 3D com o comando de software &quot;z-stack&quot; na primeira posição memorizado de acordo com o volume necessário com a ampliação escolhida. Memorize posição mais profunda em primeiro lugar, e, em seguida, a posição mais alta. Normalizar poder do laser com a profundidade. NOTA: Para múltiplos imaging campo concomitante, adquirimos 5 fatias separadas por 3 um z-passos para cada campo de adquirir um volume de 12 mm de espessura. Estas configurações podem ser adaptadas ao tipo de célula estudada. </li></ol></li><li> Aquisição de Lançamento <ol><li> Selecione o220; comando série &quot;software e escolher a duração do intervalo de tempo e do número de ciclos de tempo. Comece imagem time-lapse de acordo com o lapso de tempo e duração necessária escolhendo o comando software &quot;iniciar a experiência&quot;. NOTA: No nosso caso, nós adquirimos um volume cada 30 segundos durante 60 ciclos que representam 30 min tempo real. Menor lapso de tempo pode ser executada para controlar rolagem celular rápido no vaso sanguíneo. Neste caso, mais fina z-pilha e não mais do que dois domínios diferentes podem ser gravadas ao mesmo tempo. </li><li> O acompanhamento regular do set up. <ol><li> Sempre monitorar o animal para se certificar de que está inconsciente. NOTA: Agitação de bigodes ou pernas do animal são indicadores de avivamento. Em caso de respiração do animal é irregular, a quantidade de isoflurano pode ser ligeiramente reduzida. Deriva tecido forte durante a aquisição é um indicador do rato avivamento. </li><li> Verifique se o objetivo for devidamente imerso. Renove 37 ° C PBS between duas aquisições (30 min). NOTA: o desaparecimento progressivo de sinal durante a aquisição é indicador de PBS vazamento. </li></ol></li></ol></li><li> Fim do procedimento <ol><li> Uma vez que os vídeos exigidos sejam registrados, sacrificar o animal por deslocamento cervical rapidamente antes de reanimação. NOTA: a cicatrização de feridas, conduz à formação de uma crosta em de 24 a 48 horas o que limita a possibilidade de realizar imagem longitudinal. </li></ol></li></ol> Análise 3. Dados <ol><li> Correção de deriva <ol><li> Use software Imaris Bitplane para rastreamento automático 3D. </li><li> Abra a sequência de imagens 3D em Imaris. Selecione &quot;spot&quot; de comando e gerar rastreamento manual (clique do mouse + shift) para todos os pontos de tempo para um mínimo de quatro pontos de ancoragem diferentes, se possível homogeneamente distribuída no campo. </li><li> Aplique a correção de desvio utilizando o comando &quot;deriva correta&quot; na aba &quot;editar faixa&quot;. </li><li> Verifique a existência de tele qualidade da correcção em x, y e z, em particular para evitar a vacilação artefactual. NOTA: Se a correção não for satisfatória, tentar outros pontos de ancoragem ou excluir o vídeo da análise. </li></ol></li><li> Rastreamento de celular <ol><li> Use &quot;adicionar novos pontos de comando&quot; e selecione &quot;pontos de trilha ao longo do tempo&quot; ajuste algoritmo. Ir para a próxima etapa. </li><li> Aplicar procedimento de rastreamento automático de células, quer por todo o canal de interesse usando o comando &quot;canal de origem&quot;. Conjunto de objectos (isto é, células) de diâmetro e 10 um. Ir para a próxima etapa. NOTA: A escolha dependerá da distinção entre objectos e especificidade do sinal medido. Em suma, o rastreamento automático em todo o canal só é possível se o sinal detectado é específica para o objeto de interesse. Porque tanto SHG e ECFP são detectados pelo NDD após o filtro 485 passe curto, rastreamento será mais específico e eficiente utilizando o sinal ECFP registrado pelo NDD após o 500/550 filtros passa-banda. Agrupado ou objetos embalados densas exigirá seleção manual de objetos individuais semelhantes, para a etapa 3.1.2. </li><li> Selecione &quot;qualidade&quot; tipo de filtro, defina o limite para exclusão de rastreamento de objetos inespecíficos. Ir para a próxima etapa. </li><li> Escolha &quot;browniano Algorithm Motion&quot; com parâmetros precisos de &quot;distância Max&quot; e &quot;tamanho Gap&quot; entre dois pontos. Validar geração pista. </li><li> Uma vez que as faixas são calculadas automaticamente, selecione &quot;duração faixa&quot; tipo de filtro, definir o limite para eliminar faixas mais curto do que 120 segundos (o que representa 4 pontos de tempo). </li><li> Obrigatório: Após rastreamento automático, verificar a qualidade das faixas. <ol><li> Desenrole a barra de tempo para verificar se os objetos de seguir os caminhos de trilha. Se não, use as opções de correção ponto de trilha disponíveis na aba &quot;editar&quot;: Excluir faixas falsas ou inadequadas com &quot;excluir&quot; comando (excluir qualquer faixa point individualmente usando o comando &quot;Desligar&quot;). Se trajeto da trilha é descontínua, adicionar pontos de tempo em falta (clique do mouse + Shift), selecione todos os pontos em uma determinada faixa e usar &quot;conectar&quot; o comando na aba &quot;editar faixa&quot;. </li></ol></li></ol></li><li> Parâmetros de quantificação <ol><li> Selecione a aba &quot;Estatísticas&quot; no comando &quot;Preferências&quot;, escolha os parâmetros dinâmicos de interesse na lista. Selecione &quot;Exportar todas as estatísticas para o arquivo&quot; comando e salvar o arquivo excel gerado. Nota: Vários parâmetros dinâmicos são directamente obtidos a partir do software, tais como comprimentos de pista, velocidade instantânea, velocidade média, e acompanhar retidão. Coeficiente de motilidade 6, 20 pode ser determinada, mas não é directamente fornecida pelo software. </li><li> Para cada célula, calcular a média dos deslocamentos para cada período de tempo (dt) (para um filme de 30 segundos lapso de tempo, dt será de 30, 60, 90 s, etc.).Traça-se a média de deslocamento do quadrado como uma função do intervalo de tempo. Obter coeficiente de motilidade, calculando o declive da parte linear da curva. </li></ol></li></ol>

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The authors have nothing to disclose.

Os pontos críticos da metodologia de imagiologia in vivo, são para assegurar a estabilidade do foco, de modo a maximizar a duração das imagens e para minimizar o risco de contaminação bacteriana e inflamação, que pode ter impacto na dinâmica das células inflamatórias. Imagem da medula óssea do crânio segue estes objectivos como a cirurgia realizada para obter o acesso para a medula óssea é mínima. A utilização de materiais estéreis e anti-sépticos é essencial para limitar o risco de ...

The bone marrow plays a central role in hematopoiesis and represents the main reservoir of monocytes that constitutively recirculate between the blood and the medullar parenchyma, renew the pool of circulating monocytes with a short life span 2,3 and participate in the reconstitution of the steady state tissue-macrophages and dendritic cells 4. During inflammation or after transient aplasia, monocytes are actively mobilized from either the bone marrow or the spleen 5, 6, 7 and colonize inflamed organs. Several chemoattractant axis have been involved in the process of myeloid cell mobilization from the bone marrow 8, 5, 6,9. Beyond the myeloid compartment the bone marrow is also an important site of T lymphocyte priming 10 and a niche of immunological memory 11,12. Thus, this tissue is central for numerous investigations in the field of hematology and immunology. Our knowledge on the structural organization of medullar myeloid cells mainly arises from the analysis of histological section of fixed tissues 13. This static view does not allow for a study of the cellular exchange dynamic between the different compartments of the bone marrow, which is the basis of its functional activity.
Intravital imaging constitutes an important biological input in the study of cell mobility, cell adherence and cell-to-cell interactions, which were previously described only from in vitro systems. Technical challenges for proper intravital imaging include the ability to reach the tissue of interest in an optical point of view, and to maximize its isolation from physiological (breath, muscle or peristaltic contractions) or mechanical drifts (tissue disruption and extension following surgery, and exposure to microscope objective as well as temperature and vascular/oxygenation perturbations). Microscopic drifts may limit the ability to keep the focus a long time and could introduce artifacts in the quantification of cell motility. One alternative, validated for several tissues to reduce these technical difficulties, is to work on explanted tissue incubated in a thermostated and oxygenated medium; however, complete disruption of the lymphatic and vascular circulation may be problematic. Intravital imaging of skull bone marrow has several advantages concerning these issues. Firstly, it requires minimal surgical action. Secondly, thickness of the bone in this region allows direct visualization of bone marrow niches without abrasion, thus reducing physiological perturbations. The medullar network can be imaged in the parasagittal region of the bone; however the sinusoids are more visible in the fronto-parietal area where the bone matrix is thinner12,14. 
Intravital imaging relies on the availability of the most accurate fluorescent reporter tagging the population of interest. In vitro labelling of purified cell population before adoptive transfer led to important characterization of hematopoietic stem cell niches 15 or bone marrow endothelial microdomains favouring tumor engraftment 16, and provided several fundamental inputs on key concepts in immunology 17 . However, this approach usually requires hundreds of thousands of cells to get a chance to detect them afterwards in vivo. This could be explained by the high mortality rate following staining, the dilution in the whole body and the change in the activation state, which might lead to biased homing. Endogenous tagging from transgenic mouse system greatly overcomes these limitations and has allowed to image the behaviour of endogenous osteoblast 8, megakaryocytes 18 or myeloid-lineage subsets 6 . Nevertheless, one has to be cautious when considering the specificity of the fluorescent reporter among the studied subset. 
The Csf1r-Gal4VP16/UAS-ECFP, called MacBlue mouse 1, is a valuable transgenic system to study medullar monocytes with real time imaging 6. Intravenous injection of high molecular weight rhodamin-dextran distinguishes the medullar parenchyma from the vascular sinusoid network of the bone marrow. Using this approach, it is possible to track the monocyte behaviour in the different medullar compartments in a specific physiopathological context of interest. Furthermore, we propose an additional strategy to compare monocyte dynamics with that of neutrophils through in vivo labelling using a specific antibody.

<table><tbody><tr><td>Ketamin</td><td>Merial</td><td></td><td>100 mg/ml, anesthetic</td></tr><tr><td>Xylazin</td><td>Bayer HealthCare</td><td></td><td>10 mg/ml, anesthetic</td></tr><tr><td>Isofluran</td><td>Baxter</td><td></td><td>2.5%, anesthetic</td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt;/NO&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>70/30 mixture, anesthetic</td></tr><tr><td>Rhodamin-Dextran</td><td>Invitrogen</td><td></td><td>2 MDa, 10 mg/ml, Vascular staining</td></tr><tr><td>Ly6G-PE</td><td>Becton-Dickinson</td><td></td><td>clone 1A8, neutrophils staining</td></tr><tr><td>Stereotactic holder</td><td>Home made</td><td></td><td>surgery</td></tr><tr><td>Ethanol 70%</td><td></td><td></td><td>surgery</td></tr><tr><td>Sterile scissors and nippers</td><td></td><td></td><td>surgery</td></tr><tr><td>Rubber ring</td><td></td><td></td><td>18 mm diameter, surgery</td></tr><tr><td>Glubran 2</td><td>Queryo Medical</td><td></td><td>Surgical Glue, rubber ring fixation</td></tr><tr><td>Small gauge needles</td><td>Terumo</td><td></td><td>surgery</td></tr><tr><td>Zeiss LSM 710 NLO multiphoton microscope&amp;nbsp;</td><td>Carl Zeiss</td><td></td><td>Microscope</td></tr><tr><td>Ti:Sapphire crystal laser&amp;nbsp;</td><td>Coherent Chameleon Ultra</td><td></td><td>140 fsec pulses of NIR light</td></tr><tr><td>Zen 2010</td><td>Carl Zeiss</td><td></td><td>Acquistion Software</td></tr><tr><td>Imaris Bitplane&amp;nbsp;</td><td>Bitplane</td><td></td><td>Analysis Software, 3D automatic tracking</td></tr><tr><td>PBS 1X</td><td>D. Dutscher</td><td></td><td>surgery</td></tr><tr><td>Thermostated chamber</td><td>Carl Zeiss</td><td></td><td>intravital imaging</td></tr></tbody></table>

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.

A estrutura do crânio mouse oferece uma boa oportunidade para estudar a fisiologia da medula óssea por imagem intravital. O osso ser magro em torno da área frontal-parietal, é possível ter acesso a nichos medulares sem abrasão do osso. A Figura 1 representa um campo 2D grande do crânio de um camundongo transgênico MacBlue. A matriz do osso é composta principalmente de colagénio I facilmente detectável por SHG 19. Injecção de rodamina-dextrano mancha da rede vascular da medula ós...

Watch this Scientific Journal Video about Tracking Mouse Bone Marrow Monocytes In Vivo at JoVE.com

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 ...

tracking-mouse-bone-marrow-monocytes-in-vivo

Research

JoVE Journal

Immunology and Infection

Tracking Mouse Bone Marrow Monocytes In Vivo

9.5K visualizações.  Sorbonne Universités, UPMC Univ Paris 06, CR7. 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. 

Vídeo: Tracking Mouse Bone Marrow Monocytes In Vivo

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 ...

Imunologia e Infecção

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

Isolamento e injecção intravenosa de Murino de Medula Óssea Derivado monócitos

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Femoral Medula Óssea aspiração Live Ratos

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Medicina

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Imagiologia neutrófilos e monócitos em Mesenteric Veins por Microscopia intravital em ratos anestesiados em Tempo Real

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Modelo Câmara fêmur Janela para<em&gt; In Vivo</em&gt; Acompanhamento de celular na Murino de Medula Óssea

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Um modelo de rato In Vivo para ativação de células T Naïve CD4 medida, proliferação e diferenciação de células Th1 induzida pelas células dendríticas derivadas de medula óssea

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

Preparação de medula óssea inteira para análise de citometria de massa de células de linhagem de neutrófilos

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Biologia

<meta charset="utf8"></head><body>Injeção intrafemoral para estudo da medula óssea em camundongos vivos

Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. In murine models, this technique has been adapted to enhance the seeding efficiency of transplanted hematopoietic stem and progenitor cells (HSPCs). This paper describes a detailed step-by-step technical procedure of IF injection and the following bone marrow (BM) aspiration in mice that allows for serial characterization of cells present in the BM. This method enables the transplantation of valuable samples with low cell numbers that are particularly difficult to engraft by intravenous injection. This procedure facilitates the creation of xenografts that are critical for pathological analysis. While it is easier to access peripheral blood (PB), the cellular composition of PB does not reflect the BM, which is the niche for HSPCs. Therefore, procedures providing access to the BM compartment are essential for studying hematopoiesis. IF injection and serial BM aspiration, as described here, allow for the prospective retrieval and characterization of cells enriched in the BM, such as HSPCs, without sacrificing the mice.

<meta charset="utf8"></head><body>Injeções intrafemorais simplificadas usando camundongos vivos permitem análise contínua da medula óssea

This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone marrow.

Injeções intrafemorais simplificadas usando camundongos vivos permitem análise contínua da medula óssea

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. ...

Acompanhando Rato Medula Óssea monócitos

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