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.

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...

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

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.

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 ...

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

Research

JoVE Journal

Immunology and Infection

1.3K Views.  West Virginia University School of Medicine. 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.

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

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFN&gamma;). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFN&gamma; and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFN&gamma; and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

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).

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

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.

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.

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.