assessing phagocytic function of neonatal bone marrow-derived macrophages using a fluorescent bacterial uptake assay

Phagocytic Assay Evaluation of Neonatal Bone Marrow Derived Macrophages 

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).

Author Spotlight: Developing a Reproducible Method for Isolating Bone Marrow-Derived Macrophages from Neonatal Mice to Advance Early Life Immune Responses

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

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.

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

To begin resuspend the mouse neonatal bone marrow derived macrophages in complete DMEM without phenol red or antibiotics. Seed 500 microliters of cell suspension at a density of 200, 000 per quadrant in a 35 millimeter quad dish. Remove the precalculated stock of Escherichia coli from minus 80 degrees Celsius.Aliquot the desired volume of cell suspension into a 1.7 milliliter micro centrifuge tube to prepare bacterial inoculum at a multiplicity of infection of 25, then add PBS to a total volume of one milliliter. Centrifuge the bacterial suspension at 2000 G for five minutes and resuspend the pellet in 50 microliters of PBS. Add green fluorescent pH sensitive dye to a final concentration of 500 micro molars and incubate for 20 minutes in the dark for dye conjugation.Wash the bacteria four times with one milliliter of PBS by centrifugation at 1000 G for five minutes. Resuspend the pellet in 500 microliters of complete DMEM without phenol red and add the bone marrow derived macrophage cultures. Incubate the culture at 37 degrees Celsius and 5%carbon dioxide for four hours.Add 200 nanograms of cell permeable red fluorescent dye to stain the lysosomes, Upon bacterial infection, abundant green fluorescent bacteria phagocytosed inside bone marrow derived macrophages were detected. The green fluorescence further localizes with red fluorescence indicative of acidified lysosomes. The phagocytosis and appearance of green pH sensitive dipositive neonatal bone marrow derived macrophages were also observed throughout the four hour infection.

assessing-phagocytic-function-neonatal-bone-marrow-derived

Research

JoVE Journal

Immunology and Infection

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 ...

Non-Enzymatic Method for Isolating Neonatal Mouse Bone Marrow Cells

After decapitation, sterilize the body of the anesthetized neonate mouse with 70%ethanol. Using forceps and fine tipped surgical scissors, make an incision between the abdomen and hind legs. Then remove the skin by pulling it toward the foot of the hind limbs.Scrape the connective tissue and muscle attached to the bones using sharp edged forceps. Cut the tibia and femur with scissors. Use forceps to place the tibia and femur in a MEM culture media tube on ice.Transfer the bones to a 40 micron strainer with a collection tube. Crush the bones with a three milliliter syringe plunger against the strainer to release the marrow. Centrifuge the cell suspension at 350 G for five minutes at four degrees Celsius.Aspirate the supernatant and gently re-suspend the cells in 0.2%sodium chloride to make a hypotonic solution for the lysis of erythrocytes. Immediately add an equal volume of 1.6%sodium chloride to make the solution isotonic. Centrifuge the supernatant and remove the lysis solution.Re-suspend the cells in complete DMEM and count the cells on an automated cell counter.

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

To begin, seed 20 million neonate mouse bone marrow cells per T75 flask in 10 milliliters of complete DMEM containing 2.5 milliliters of 10%L929 cell supernatant. Incubate the culture flask at 37 degrees Celsius with 5%carbon dioxide for five days. On day three of differentiation, add two milliliters of L929 conditioned media into the flask.Under an inverted microscope with 20x magnification, daily observe the cells. To harvest the macrophages on day five, aspirate the differentiation media from the flask. Wash the cells with five milliliters of PBS to remove non-adherent cells and serum proteins.Add five milliliters of 0.05%trypsin EDTA to the flask and incubate at 37 degrees Celsius incubator with 5%carbon dioxide. After five minutes, add five milliliters of DMEM and pipette to detach the cells. Centrifuge the cell suspension at 350 g for five minutes.Dissociate the cell palate in one milliliter of complete DMEM. Count the cells on an automated cell counter. In the presence of L929 cell supernatant, bone marrow cells were differentiated into macrophages within five days.Spindle-shaped cell formation was observed from the second day with nearly half displaying this morphology by day three and a majority by day five.