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...

Phagocytic Assay Evaluation of Neonatal Bone Marrow Derived Macrophages 

<ol>
	<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. M., Davis, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+and+opportunities+to+enhance+global+drug+development+in+neonates.">Challenges and opportunities to enhance global drug development in neonates.</a> Curr Opin Pediatr. 29 (2), 149-152 (2017).</li><li>Lagler, F. B., Hirschfeld, S., Kindblom, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+clinical+trials+for+children+and+young+people.">Challenges in clinical trials for children and young people.</a> Arch Dis Child. 106 (4), 321-325 (2021).</li><li>Heap, R. E., 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=Proteomics+characterization+of+the+L929+cell+supernatant+and+its+role+in+BMDM+differentiation.">Proteomics characterization of the L929 cell supernatant and its role in BMDM differentiation.</a> Life Sci Alliance. 4 (6), 202000957 (2021).</li><li>Weischenfeldt, J., Bone Porse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=marrow-derived+macrophages+(BMM):+Isolation+and+Applications.">marrow-derived macrophages (BMM): Isolation and Applications.</a> CSH Protoc. 2008, (2008).</li><li>Goncalves, R., Kaliff Teofilo Murta, G., Aparecidade de Souza, I., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+bone+marrow-derived+macrophages+from+mice.">Isolation and culture of bone marrow-derived macrophages from mice.</a> J Vis Exp. (196), e64566 (2023).</li><li>Perfetto, S. P., 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=Amine+reactive+dyes:+An+effective+tool+to+discriminate+live+and+dead+cells+in+polychromatic+flow+cytometry.">Amine reactive dyes: An effective tool to discriminate live and dead cells in polychromatic flow cytometry.</a> J Immunol Methods. 313 (1-2), 199-208 (2006).</li><li>Springer, T., Galfre, G., Secher, D. S., Milstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mac-1:+A+macrophage+differentiation+antigen+identified+by+monoclonal+antibody.">Mac-1: A macrophage differentiation antigen identified by monoclonal antibody.</a> Eur J Immunol. 9 (4), 301-306 (1979).</li><li>Austyn, J. M., Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F4/80,+a+monoclonal+antibody+directed+specifically+against+the+mouse+macrophage.">F4/80, a monoclonal antibody directed specifically against the mouse macrophage.</a> Eur J Immunol. 11 (10), 805-815 (1981).</li><li>Akbarshahi, H., Menzel, M., Posaric Bauden, M., Rosendahl, A., Andersson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+of+murine+CD68++CCR2++and+CD68++CD206++lung+macrophages+in+acute+pancreatitis-associated+acute+lung+injury.">Enrichment of murine CD68+ CCR2+ and CD68+ CD206+ lung macrophages in acute pancreatitis-associated acute lung injury.</a> PLoS One. 7 (10), e42654 (2012).</li><li>Yao, Y., Xu, X. H., Jin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+polarization+in+physiological+and+pathological+pregnancy.">Macrophage polarization in physiological and pathological pregnancy.</a> Front Immunol. 10, 792 (2019).</li><li>Lu, L., 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=Differential+expression+of+CD11c+defines+two+types+of+tissue-resident+macrophages+with+different+origins+in+steady-state+salivary+glands.">Differential expression of CD11c defines two types of tissue-resident macrophages with different origins in steady-state salivary glands.</a> Sci Rep. 12 (1), 931 (2022).</li><li>Harbeson, D., Ben-Othman, R., Amenyogbe, N., Kollmann, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outgrowing+the+immaturity+myth:+The+cost+of+defending+from+neonatal+infectious+disease.">Outgrowing the immaturity myth: The cost of defending from neonatal infectious disease.</a> Front Immunol. 9, 1077 (2018).</li><li>Kollmann, T. R., Kampmann, B., Mazmanian, S. K., Marchant, A., Levy, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protecting+the+newborn+and+young+infant+from+infectious+diseases:+Lessons+from+immune+ontogeny.">Protecting the newborn and young infant from infectious diseases: Lessons from immune ontogeny.</a> Immunity. 46 (3), 350-363 (2017).</li><li>Loopmans, S., Stockmans, I., Carmeliet, G., Stegen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+in+vitro+characterization+of+murine+young-adult+long+bone+skeletal+progenitors.">Isolation and in vitro characterization of murine young-adult long bone skeletal progenitors.</a> Front Endocrinol (Lausanne). 13, 930358 (2022).</li><li>Winterberg, T., 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=Distinct+phenotypic+features+of+neonatal+murine+macrophages).">Distinct phenotypic features of neonatal murine macrophages).</a> Eur J Immunol. 45 (1), 214-224 (2015).</li><li>de Brito Monteiro, L., 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=M-CSF-+and+L929-derived+macrophages+present+distinct+metabolic+profiles+with+similar+inflammatory+outcomes.">M-CSF- and L929-derived macrophages present distinct metabolic profiles with similar inflammatory outcomes.</a> Immunobiology. 225 (3), 151935 (2020).</li></ol>

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

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.

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.

820 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.

Phagocytic Assay Evaluation of Neonatal Bone Marrow Derived Macrophages

Summary