non-enzymatic method for isolating neonatal mouse bone marrow cells

Avaliação de ensaio fagocítico de macrófagos derivados da medula óssea neonatal 

Bone marrow encloses both hematopoietic and mesenchymal stem cell populations that are self-renewable and can be differentiated into various cell lineages.&#160;Hematopoietic stem cells in the bone marrow give rise to myeloid and lymphoid lineages1. Mesenchymal stem cells produce osteoblasts (bone), adipocytes (fat), or chondrocytes (cartilage)2. These cells have multiple applications in the field of cell biology and tissue engineering, including gene therapy3,4. Progenitor cells present in the bone marrow differentiate into specific cell types in the presence of lineage-specific growth factors. Erythropoietin promotes the proliferation of erythroid progenitor cells, granulocyte colony-stimulating factor (G-CSF) stimulates the growth of neutrophil colonies, and thrombopoietin regulates the production of platelets as a few examples of lineage-specific growth factors5. Cell surface antigen labeled FACS and magnetic-activated cell sorting (MACS) are well-established methods for isolation and purification of the specific bone marrow-derived cell types6.
Though neonatal studies are advancing toward finding the causes of neonatal deaths and addressing the complications during premature births, direct therapeutic development remains an unmet medical need. Smith and Davis stated, &#34;Pediatric patients remain therapeutic orphans&#34;7. There are several challenges, such as small samples, lifelong effects of the outcome, and ethical issues in obtaining consent in clinical studies of neonates8. Hence, there is a high demand for in vivo and in vitro study models specific to neonates to achieve translational relevance. Because of the similarities between anatomical and tissue levels, short gestational periods, and litter sizes, rodents are the most studied mammalian model system.
Here, we describe a detailed, highly feasible, and reproducible procedure for isolating bone marrow from 7-9-day-old mouse pups and their ability to differentiate into macrophages. However, a variety of cell lineages could be achieved with the use of distinct differentiation signals. We also demonstrate the presence of cell surface markers and the presence of in vitro phagocytic activity expected for bone marrow-derived macrophages (BMDMs).

Autor em destaque: Desenvolvendo um método reprodutível para isolar macrófagos derivados da medula óssea de camundongos neonatais para avançar nas respostas imunes no início da vida

Isolamento da medula óssea de camundongos neonatais e preparação de macrófagos derivados da medula óssea

Método não enzimático para isolar células da medula óssea de camundongos neonatais

Após a decapitação, esterilize o corpo do camundongo recém-nascido anestesiado com etanol a 70%. Usando fórceps e tesouras cirúrgicas de ponta fina, faça uma incisão entre o abdômen e as patas traseiras. Em seguida, remova a pele puxando-a em direção ao pé dos membros posteriores.Raspe o tecido conjuntivo e o músculo preso aos ossos usando uma pinça afiada. Corte a tíbia e o fêmur com uma tesoura. Use uma pinça para colocar a tíbia e o fêmur em um tubo de meio de cultura MEM no gelo.Transfira os ossos para uma peneira de 40 mícrons com um tubo de coleta. Esmague os ossos com um êmbolo de seringa de três mililitros contra a peneira para liberar a medula. Centrifugar a suspensão celular a 350 G durante cinco minutos a quatro graus Celsius.Aspire o sobrenadante e ressuspenda suavemente as células em cloreto de sódio a 0,2% para fazer uma solução hipotônica para a lise dos eritrócitos. Adicione imediatamente um volume igual de cloreto de sódio a 1,6% para tornar a solução isotônica. Centrifugar o sobrenadante e remover a solução de lise.Ressuspenda as células no DMEM completo e conte as células em um contador de células automatizado.

non-enzymatic-method-for-isolating-neonatal-mouse-bone-marrow-cells

Research

JoVE Journal

Immunology and Infection

Non-Enzymatic Method for Isolating Neonatal Mouse Bone Marrow Cells

93 visualizações. Após a decapitação, esterilize o corpo do camundongo recém-nascido anestesiado com etanol a 70%. Usando fórceps e tesouras cirúrgicas de ponta fina, faça uma incisão entre o abdômen e as patas traseiras. Em seguida, remova a pele puxando-a em direção ao pé dos membros posteriores.Raspe o tecido conjuntivo e o músculo preso aos ossos usando uma pinça afiada. Corte a tíbia e o fêmur com uma tesoura. Use uma pinça para colocar a tíbia e o fêmur em um tubo de meio de cul...

Vídeo: Método não enzimático para isolar células da medula óssea de camundongos neonatais

Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging and time-consuming, yet for some models, it is translationally relevant and necessary. This protocol describes an efficient and straightforward method for preparing bone marrow cells from 7-9-day-old pups. These cells can then be further isolated or differentiated into specific cell types of interest. Macrophages are crucial immune cells that play a major role in inflammation and infection. During development, neonatal macrophages contribute significantly to tissue remodeling. Moreover, the phenotype and functions of neonatal macrophages differ from those of their adult counterparts. This protocol also outlines the differentiation of neonatal macrophages from the isolated bone marrow cells in the presence of L929-conditioned medium. Surface markers for differentiated neonatal macrophages were assessed using flow cytometric analysis. To demonstrate functionality, the phagocytic efficiency was also tested using pH-sensitive dye-conjugated Escherichia coli.

This protocol describes a non-enzymatic and straightforward method for isolating 7-9-day-old neonatal mouse bone marrow cells and generating differentiated macrophages using a supernatant of L929 cells as a source of granulocyte colony-stimulating factor (M-CSF). The bone marrow-derived macrophages were further analyzed for surface antigens F4/80, CD206, CD11b, and functional competency.

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging ...