non-enzymatic method for isolating neonatal mouse bone marrow cells

Evaluación del ensayo fagocítico de macrófagos derivados de la médula ósea 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 destacado: Desarrollo de un método reproducible para aislar macrófagos derivados de la médula ósea de ratones neonatos para avanzar en las respuestas inmunitarias tempranas de la vida

Aislamiento de médula ósea de ratón neonatal y preparación de macrófagos derivados de la médula ósea

The scope of this study is to understand some of the limitations in the early life immune response to bacterial infection. In order to accomplish this, we need to develop and use research models that are specific to neonates. Isolation of bone marrow from a day-seven-old-pup is challenging and not well established.Neonatal bone marrow derived macrophages are sensitive and prone to stress when compared to other BMDM. The technique used in this protocol is simple and reproducible. This method guides the users in isolating bone marrow from seven to nine-day-old neonatal mice without the use of any enzymes.This study provides a standardized strategy to generate bone marrow-derived macrophages from neonatal mice that can be used to address questions about neonatal immunology. This reproducible method provides an age-specific model for progressing neonatal immune research across many areas of expertise.

Método no enzimático para aislar células neonatales de médula ósea de ratón

Después de la decapitación, esterilizar el cuerpo del ratón neonato anestesiado con etanol al 70%. Con fórceps y tijeras quirúrgicas de punta fina, haga una incisión entre el abdomen y las patas traseras. A continuación, retira la piel tirando de ella hacia el pie de las extremidades traseras.Raspe el tejido conectivo y el músculo unido a los huesos con pinzas afiladas. Corta la tibia y el fémur con unas tijeras. Use fórceps para colocar la tibia y el fémur en un tubo de medio de cultivo MEM sobre hielo.Transfiera los huesos a un colador de 40 micras con un tubo de recolección. Triture los huesos con un émbolo de jeringa de tres mililitros contra el colador para liberar la médula. Centrifugar la suspensión celular a 350 G durante cinco minutos a cuatro grados centígrados.Aspire el sobrenadante y vuelva a suspender suavemente las células en cloruro de sodio al 0,2% para hacer una solución hipotónica para la lisis de los eritrocitos. Agregue inmediatamente un volumen igual de cloruro de sodio al 1,6% para hacer que la solución sea isotónica. Centrifugar el sobrenadante y retirar la solución de lisis.Vuelva a suspender las células en DMEM completo y cuente las células en un contador de células automatizado.

Research

JoVE Journal

Immunology and Infection

Non-Enzymatic Method for Isolating Neonatal Mouse Bone Marrow Cells

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 ...

Investigación

Inmunología e Infección

Differentiation of Neonatal Bone Marrow-Derived Macrophages Using a Supernatant of L929 Cells

Diferenciación de macrófagos derivados de la médula ósea neonatal utilizando un sobrenadante de células L929

Assessing Phagocytic Function of Neonatal Bone Marrow-Derived Macrophages Using a Fluorescent Bacterial Uptake Assay

Evaluación de la función fagocítica de los macrófagos derivados de la médula ósea neonatal mediante un ensayo de captación bacteriana fluorescente