This work was supported by the National Institutes of Health [R01 AI163333] to CMR. We acknowledge additional funding support provided to the West Virginia University Flow Cytometry and Single Cell Core Facility by the following grants: WV CTSI grant GM104942, Tumor Microenvironment CoBRE grant GM121322 and NIH grant OD016165.

All procedures were approved by the West Virginia Institutional Animal Care and Use Committees and were performed following the recommendations of the Guide for the Care and Use of Laboratory Animals by the National Research Council. C57BL/6J mouse pups were used for this study. The details of all the reagents and equipment used are listed in the Table of Materials.
1. Media preparation
<ol>
	<li>Prepare 3 mL of MEM culture media supplemented with 10% FBS, 2 mM glutamine, 25 mM HEPES, and penicillin (100 U/mL)/streptomycin (100 &#181;g/mL) in a 5 mL centrifuge tube, and keep it on ice.</li>
	<li>If needed for storing bone marrow progenitors in liquid nitrogen, prepare freezing media containing 10% DMEM, 80% FBS, and 10% DMSO.</li>
	<li>For complete DMEM, prepare DMEM supplemented with 10% FBS, 2 mM glutamine, 25 mM HEPES, and penicillin (100 U/mL)/streptomycin (100 &#181;g/mL).</li>
	<li>For macrophage differentiation, prepare DMEM supplemented with 10% FBS, 2 mM glutamine, penicillin (100 U/mL)/streptomycin (100 &#181;g/mL), and 10% L929-cell supernatant9,10,11.</li>
</ol>
2. L929-cell supernatant preparation
<ol>
	<li>Thaw L929 cells stored in liquid nitrogen and transfer to a 15 mL centrifuge tube containing 5 mL of complete DMEM. Centrifuge at 350 x g for 5 min at room temperature.</li>
	<li>Resuspend the cells in 5 mL of complete DMEM media and seed them into a T25 tissue culture flask at the density of 2&#215;105 cells. Count the cells with an automated cell counter using 0.4% trypan blue. Incubate at 37 &#176;C with 5% CO2 until they reach 70% confluency.</li>
	<li>To detach the cells, first wash them with 5 mL of phosphate-buffered saline (PBS). Aspirate the PBS and replace it with 2.5 mL 0.05% trypsin-EDTA.</li>
	<li>Incubate at 37 &#176;C with 5% CO2 for 5 min and add 5 mL of complete DMEM.</li>
	<li>Collect the cells and centrifuge at 350 x g for 5 min at room temperature.</li>
	<li>Resuspend the cell pellet in 15 mL of complete DMEM media and transfer it to a T75 flask.</li>
	<li>Incubate at 37 &#176;C with 5% CO2 until the cells reach 90% confluency, and then collect the medium and centrifuge at 500 x g for 5 min at 4 &#176;C. Filter the media containing M-CSF through a 0.45 &#181;m filter and freeze at -80 &#176;C in 5 mL aliquots.</li>
</ol>
3. Animal preparation
<ol>
	<li>Under a biosafety cabinet, carefully separate the 7-9-day old C57BL/6J mouse pups (a litter of 6 pups) from the dam and place them in a separate cage.</li>
	<li>Soak a cotton ball in 2 mL of veterinary-grade isoflurane in a bell jar or other containment chamber.</li>
	<li>Place a pup in the chamber and close the lid. Monitor the neonate for approximately 90 s to ensure it becomes motionless and unconscious.</li>
	<li>Quickly remove the pup and decapitate it with sharp scissors (following institutionally approved protocols) before the pup can regain consciousness. 
	NOTE: Neonates do not breathe sufficiently deep for euthanasia by isoflurane inhalation alone.</li>
	<li>Ensure to close the lid of the container after each pup is removed. The same cotton ball can be used for 5-7 pups.</li>
</ol>
4. Isolation of neonatal bone marrow
<ol>
	<li>Sterilize the body of the neonate with 70% ethanol.</li>
	<li>&#8220;Using forceps and fine-tipped&#160;surgical scissors, make an incision between the 
	abdomen and hind legs.&#160;Remove the skin by pulling toward the foot of the hind limbs.</li>
	<li>Scrape the connective tissue and muscle attached to the bones using sharp-edged forceps.</li>
	<li>Cut the tibia and femur, as&#160;shown in Figure 1, with scissors to detach and&#160;remove.&#160;Using forceps, place these bones in the media tube on ice. Repeat the process for each pup.</li>
	<li>Transfer the bones with the help of forceps to a 40 &#181;m strainer with a collection tube. Crush the bones and release the marrow by pressing with a 3 mL syringe plunger against the strainer.</li>
	<li>Centrifuge the cell suspension at 350 x g for 5 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant and resuspend the cells by gentle pipetting in 0.2% NaCl, a hypotonic solution for the lysis of erythrocytes. Immediately add an equal volume of 1.6% NaCl to bring the solution to isotonic.</li>
	<li>Centrifuge at 350 x g for 5 min at 4 &#176;C and remove the lysis solution.</li>
	<li>Resuspend in complete DMEM for immediate use or freezing media for storage and count the cells using a hemocytometer or automated cell counter. 
	NOTE: For the present study, this method yielded 6.55 (&#177; 1.44) &#215; 106 bone marrow cells/pup (Figure 1E).</li>
	<li>Store the cells at -80 &#176;C in freezing media or use them immediately as described below. 
	NOTE: Because of the age, bones are transparent and clear enough to visualize the marrow through them. It is important to be careful while pulling the bones as the application of slight pressure on them may be enough to release the marrow. The marrow in the neonatal bones is in the form of liquid rather than an intact thread found in adult bones. It is difficult to collect the marrow if it escapes during the collection of the bones.</li>
</ol>
5. Differentiation of neonatal bone marrow-derived macrophages
<ol>
	<li>Seed 2 &#215; 107 bone marrow cells per T75 flask in 10 mL of complete DMEM that contains 10% L929-cell supernatant (2.5 mL) as a source of M-CSF. Incubate the culture dishes at 37 &#176;C with 5% CO2 for 5 days. Add 2 mL of L929-conditioned media on day 3 of differentiation.</li>
	<li>Observe the cells under the microscope daily using an inverted microscope and imaging system with 20x magnification. Upon differentiation, macrophages should appear adherent, elongated, and heterogenous (Figure 2).</li>
	<li>To harvest the macrophages for downstream assays, aspirate the differentiation media.</li>
	<li>Wash the cells with 5 mL of PBS to remove non-adherent cells and serum proteins that will interfere with detachment. Aspirate the PBS.</li>
	<li>Harvest the cells by adding 5 mL of 0.05% trypsin-EDTA to the flask. Place the culture flask at 37 &#176;C with 5% CO2 incubator for 5 min and add 5 mL of DMEM.</li>
	<li>Pipette the cells to detach and centrifuge at 350 x g for 5 min at room temperature.</li>
	<li>Dissociate the cell pellet in 1 mL of complete DMEM, and count and check the cell viability using trypan blue. BMDMs isolated using this method are 7.05 (&#177; 2.52) &#215;105 cells/pup (Figure 1E). 
	NOTE: Observe the presence of spindle-shaped cells that begin to appear on day two of differentiation (Figure 1B, red arrows). If the color of the media turns yellow, indicating it is spent, add more L929-conditioned media on day 4 of differentiation; complete replacement of media is not recommended.</li>
</ol>
6. Immunolabeling and flow cytometric analysis
<ol>
	<li>Block non-specific antibody labeling of the BMDMs (2x105/label) by treating with 10 &#181;L/107 cells of FcR blocking reagent according to manufacturer recommendations in a volume of 100 &#956;L/label flow cytometry buffer (PBS supplemented with 2 mM EDTA and 0.5% bovine serum albumin). Keep on ice for 10 min. 
	NOTE: The cells can be blocked in bulk or in individual wells or tubes. All subsequent steps indicate amounts and volumes per individual cell marker label at a final volume of 100 &#956;L.</li>
	<li>Wash the cells by adding 200 &#181;L of flow cytometry buffer and centrifuge at 525 x g for 7 min at room temperature.</li>
	<li>Remove the supernatant and seed the cells in a U-bottom 96 well plate at a density of 5 &#215; 105 cells per well. Stain cells by adding FVS780-Live/Dead cell-counting dye diluted to 3,000-fold and 0.625 &#181;L of BV786-CD11b, PE-F4/80, and AlexaFluor488-CD206 in 100 &#181;L of flow cytometry buffer either individually or in combination12,13,14,15. Incubate the cells for 45 min on ice covered with foil.</li>
	<li>Add 100 &#181;L of flow cytometry buffer and wash the cells by centrifuging at 525 x g for 7 min at room temperature.</li>
	<li>Aspirate the supernatant and vortex the cell pellet gently. Add 100 &#181;L of 0.4% paraformaldehyde to each well and incubate overnight at 4 &#176;C.</li>
	<li>Wash the cells by adding 100 &#181;L of flow cytometry buffer and pellet by centrifugation at 525 x g for 7 min at room temperature.</li>
	<li>Resuspend the cells in 400 &#181;L of flow cytometry buffer and analyze using a flow cytometer.</li>
</ol>
7. In vitro assay to evaluate the phagocytic efficacy of neonatal bone marrow-derived macrophages
<ol>
	<li>Resuspend the BMDMs in complete DMEM without phenol red or antibiotics and seed them at a density of 2 &#215; 105/ quadrant in a 35 mm quad dish at a volume of 500 &#181;L.</li>
	<li>To prepare the bacterial inoculum at a multiplicity of infection (MOI) of 25 or other desired MOI, remove a pre-calculated stock of Escherichia coli O1:K1:H7 stored at -80 &#176;C.</li>
	<li>Aliquot the desired volume of bacteria into a 1.7 mL microcentrifuge tube. Wash the bacteria by adding PBS to a total volume of 1 mL. Pellet the bacteria by centrifugation at 2,000 x g for 5 min at room temperature. Repeat the washing step.</li>
	<li>Resuspend the bacterial pellet in 50 &#181;L of PBS and add green fluorescent pH-sensitive dye to a final concentration of 500 &#181;M. This is a pH-sensitive dye with no fluorescent signal at neutral pH and fluoresces brightly in acidic environments, such as acidified phagosomes, during the process of phagocytosis.</li>
	<li>Incubate the bacterial cells for 20 min in the dark for dye conjugation.</li>
	<li>Wash the bacteria four times with 1 mL of PBS by centrifugation at 1000 &#215; g for 5 min at room temperature.</li>
	<li>Resuspend the bacteria in 500 &#181;L of complete DMEM without phenol red and add to the BMDM cultures.</li>
	<li>Incubate the BMDMs at 37 &#176;C in 5% CO2 incubator for 4 h and then add 200 ng of a cell-permeable red fluorescent dye that stains the lysosomes. 
	NOTE: Phagocytic bacteria can be visualized by fluorescent microscopy.</li>
</ol>

Phagocytic Assay Evaluation of Neonatal Bone Marrow Derived Macrophages 

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	<li>Lucas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+organization+of+the+bone+marrow+and+its+role+in+hematopoiesis.">Structural organization of the bone marrow and its role in hematopoiesis.</a> Curr Opin Hematol. 28 (1), 36-42 (2021).</li><li>Deb, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+stem+cells+turn+into+bone+and+fat.">How stem cells turn into bone and fat.</a> N Engl J Med. 380 (23), 2268-2270 (2019).</li><li>Lin, H., Sohn, J., Shen, H., Langhans, M. T., Tuan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+mesenchymal+stem+cells:+Aging+and+tissue+engineering+applications+to+enhance+bone+healing.">Bone marrow mesenchymal stem cells: Aging and tissue engineering applications to enhance bone healing.</a> Biomaterials. 203, 96-110 (2019).</li><li>Soleimani, M., Nadri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+isolation+and+culture+of+mesenchymal+stem+cells+from+mouse+bone+marrow.">A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow.</a> Nat Protoc. 4 (1), 102-106 (2009).</li><li>Kaushansky, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage-specific+hematopoietic+growth+factors.">Lineage-specific hematopoietic growth factors.</a> N Engl J Med. 354 (19), 2034-2045 (2006).</li><li>Huang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+protocol+for+isolation+and+culture+of+mesenchymal+stem+cells+from+mouse+bone+marrow.">An improved protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow.</a> J Orthop Translat. 3 (1), 26-33 (2015).</li><li>Smith, A. 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The authors have no conflicts of interest relevant to this article.

Research involving neonatal mouse models can present a number of challenges. Neonates have a developing immune system that is unique compared to adults8. As such, data generated from adult animal models should not be assumed to apply to newborns, and several published works have articulated this idea well18,19. Therefore, neonatal-specific models and sources of cells are necessary to study the intricacies of the early-life immune response....

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).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>40 &#181;m strainer</td>
			<td>Greiner</td>
			<td>542040</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>96 well round (U) bottom plate</td>
			<td>Thermo Scientific</td>
			<td>12-565-65</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11b-BV786</td>
			<td>BD Biosciences</td>
			<td>740861</td>
			<td>FACS analysis</td>
		</tr>
		<tr>
			<td>Anti-mouse CD206-Alexa Fluor488</td>
			<td>BD Biosciences</td>
			<td>141709</td>
			<td>FACS analysis</td>
		</tr>
		<tr>
			<td>Anti-mouse F4/80-PE</td>
			<td>BD Biosciences</td>
			<td>565410</td>
			<td>FACS analysis</td>
		</tr>
		<tr>
			<td>Countess3</td>
			<td>Thermo Scientific</td>
			<td>TSI-C3ACC</td>
			<td>Automated cell counter</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Hyclone</td>
			<td>SH30022.01</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>VWR</td>
			<td>WN182</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>DPBS, 1x</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Escherichia coli O1:K1:H7</td>
			<td>ATCC</td>
			<td>11775</td>
			<td>Infection</td>
		</tr>
		<tr>
			<td>EVOS FL&#160;</td>
			<td>Invitrogen</td>
			<td>12-563-649</td>
			<td>Cell Imaging System&#160;</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Avantor&#160;</td>
			<td>76419-584</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>FluoroBright BMDM</td>
			<td>Thermo fisher Scientific</td>
			<td>A1896701</td>
			<td>Dye free culture media</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Cytiva</td>
			<td>SH30034.01</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Cytiva</td>
			<td>SH30237.01</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>L-929</td>
			<td>ATCC</td>
			<td></td>
			<td>Differentiation</td>
		</tr>
		<tr>
			<td>LSRFortessa</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td>Flowcytometer</td>
		</tr>
		<tr>
			<td>Lysotracker red DND 99</td>
			<td>Invitrogen</td>
			<td>L7528</td>
			<td>Fluorescent dye</td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>Corning</td>
			<td>15-010-CV</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Penicillin /streptomycin&#160;</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>pHrodo green STP ester&#160;</td>
			<td>Invitrogen</td>
			<td>P35369</td>
			<td>Fluorescent dye</td>
		</tr>
		<tr>
			<td>T75 flask</td>
			<td>Cell star</td>
			<td>658170</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300120</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Zeiss 710&#160;</td>
			<td>Zeiss</td>
			<td>P20GM103434</td>
			<td>Confocal</td>
		</tr>
	</tbody>
</table>

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.

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.

Using the method outlined in this study, 25 to 37 million bone marrow cells can be successfully isolated from a litter size of five C57BL/6 mouse pups. This method has been validated with litter sizes ranging from 5 to 7 pups. The minimum age for isolation in our experiments has been 7 days old. Depending on the litter size and the number of cells required for the experiment being less than a million, researchers could attempt this protocol for mice younger than 7 days old. In the presence of L929-cell supernatant as a s...

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 ...

neonatal-mouse-bone-marrow-isolation-preparation-bone-marrow-derived

Research

JoVE Journal

Immunology and Infection

970 Views.  West Virginia University School of Medicine. 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 m...

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