Name: generation of large numbers of myeloid progenitors and dendritic cell precursors from murine bone marrow using a novel cell sorting strategy
Uploaded: 2018-08-10T13:30:00
Description: Watch this Scientific Journal Video about Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy at JoVE.com

We are grateful for technical assistance from Alison Church Bird at the Auburn University School of Veterinary Medicine Flow Cytometry Facility, for funding from the NIH to EHS R15 R15 AI107773 and to the Cellular and Molecular Biology Program at Auburn University for summer research funding to PBR.

All animal work was approved by the Auburn University Institutional Animal Care and Use Committee in accordance with the recommendations outlined in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.
1. Preparation for Bone Marrow Collection
<ol>
	<li>Prepare 250 mL complete media by adding a solution of Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, and 50 &#181;M 2-mercaptoethanol to the top...

<ol>
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G., Hiltbold Schwartz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+developmental+stages,+kinetics,+and+phenotypes+exhibited+by+myeloid+cells+driven+by+GM-CSF+in+vitro.">Analysis of the developmental stages, kinetics, and phenotypes exhibited by myeloid cells driven by GM-CSF in vitro.</a> PLoS One. 12, e0181985 (2017).</li><li>Helft, J., 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=GM-CSF+Mouse+Bone+Marrow+Cultures+Comprise+a+Heterogeneous+Population+of+CD11c(+)MHCII(+)+Macrophages+and+Dendritic+Cells.">GM-CSF Mouse Bone Marrow Cultures Comprise a Heterogeneous Population of CD11c(+)MHCII(+) Macrophages and Dendritic Cells.</a> Immunity. 42, 1197-1211 (2015).</li><li>Alloatti, A., Kotsias, F., Magalhaes, J. G., Amigorena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+maturation+and+cross-presentation:+timing+matters!.">Dendritic cell maturation and cross-presentation: timing matters!.</a> Immunol Rev. 272, 97-108 (2016).</li><li>Jakubzick, C. V., Randolph, G. J., Henson, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+differentiation+and+antigen-presenting+functions.">Monocyte differentiation and antigen-presenting functions.</a> Nat Rev Immunol. 17, 349-362 (2017).</li><li>Mbongue, J. C., Nieves, H. A., Torrez, T. W., Langridge, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Dendritic+Cell+Maturation+in+the+Induction+of+Insulin-Dependent+Diabetes+Mellitus.">The Role of Dendritic Cell Maturation in the Induction of Insulin-Dependent Diabetes Mellitus.</a> Frontiers in immunology. 8, 327 (2017).</li><li>Shortman, K., Naik, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steady-state+and+inflammatory+dendritic-cell+development.">Steady-state and inflammatory dendritic-cell development.</a> Nat Rev Immunol. 7, 19-30 (2007).</li><li>Xu, Y., Zhan, Y., Lew, A. M., Naik, S. H., Kershaw, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+development+of+murine+dendritic+cells+by+GM-CSF+versus+Flt3+ligand+has+implications+for+inflammation+and+trafficking.">Differential development of murine dendritic cells by GM-CSF versus Flt3 ligand has implications for inflammation and trafficking.</a> J Immunol. 179, 7577-7584 (2007).</li><li>Naik, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+large+numbers+of+pro-DCs+and+pre-DCs+in+vitro.">Generation of large numbers of pro-DCs and pre-DCs in vitro.</a> Methods Mol Biol. 595, 177-186 (2010).</li><li>Nikolic, T., de Bruijn, M. F., Lutz, M. B., Leenen, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+stages+of+myeloid+dendritic+cells+in+mouse+bone+marrow.">Developmental stages of myeloid dendritic cells in mouse bone marrow.</a> Int Immunol. 15, 515-524 (2003).</li><li>Hettinger, J., 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=Origin+of+monocytes+and+macrophages+in+a+committed+progenitor.">Origin of monocytes and macrophages in a committed progenitor.</a> Nat Immunol. 14, 821-830 (2013).</li><li>Fogg, D. K., 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=A+clonogenic+bone+marrow+progenitor+specific+for+macrophages+and+dendritic+cells.">A clonogenic bone marrow progenitor specific for macrophages and dendritic cells.</a> Science. 311, 83-87 (2006).</li><li>Traver, D., 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=Development+of+CD8{alpha}-Positive+Dendritic+Cells+from+a+Common+Myeloid+Progenitor.">Development of CD8{alpha}-Positive Dendritic Cells from a Common Myeloid Progenitor.</a> Science. 290, 2152-2154 (2000).</li></ol>

This protocol facilitates isolation of GM-CSF-driven progenitor and precursor cell types in numbers sufficient for several types of analyses including biochemical assays, assays of cellular function in vitro, or instillation in vivo. This method represents a significant advance in the field of monocyte-derived dendritic cell development, enabling the reliable isolation and identification of cells early in this pathway of development as well as those differentiated cell types more commonly isolated in pr...

Culturing of murine bone marrow cells with the cytokine Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is widely used as a method to generate monocyte-derived dendritic cells (moDC; also known as inflammatory DC) in large numbers 1,2,3,4,5. These cells have been extremely useful in a variety of studies of dendritic cell (DC) function 6,7,8. Typically, these murine bone marrow cells are cultured for 6-8 days and are then used for study of dendritic cell function 5. These cultures had long been considered mostly homogenous, consisting of a majority of differentiated moDC. More recently, it has become clear that at the end of this 6&#8211;8 day culture period, there are indeed many moDC, as well as a large subset of differentiated monocyte-derived macrophages (moMacs) 9,10,11. Our own studies have further extended these findings demonstrating that other subsets of less developed cells, such as moDC precursors (moDP) and monocytes, remain in the cultures at low frequency even after 7 days 10. Thus, studies of dendritic cells (DC) function using cells generated by this system could reflect the responses of a broader cohort of cell types than previously appreciated.
We have learned a great deal from the study of GM-CSF-generated moDC relating to the function of these cells in the final stages of differentiation&#160;12,13,14. However, we understand significantly less about the developmental pathway of these cells&#160;2,15,16 and of how and when they exhibit specific functions such as: responsiveness to Pathogen Associated Molecular Patterns (PAMPs), phagocytosis, antigen processing and presentation&#160;13, and anti-bacterial activity. A protocol for isolation of large numbers of conventional Flt3L-driven DC progenitors and precursors has been reported&#160;17. Isolation of these distinct populations was achieved using carboxyfluorescein succinimidyl ester (CFSE)-stained bone marrow cells (to track dividing cells) and culture in Flt3L for 3 days. Cells were then depleted of linage positive cells and sorted into progenitor and precursor populations based on CD11c expression&#160;17. Another approach by Leenen&#39;s group to identify early progenitors of DC in GM-CSF-driven culture was to sort cells based on CD31 and Ly6C 18. The initial goal was to create a similar method for obtaining progenitors and precursors of GM-CSF-driven moDC. Due to the specific cell types generated by GM-CSF, we adapted the approach and sorting strategy based on expression of molecules that were expressed at early and later stages of development. We ultimately determined that Ly6C, CD115 (CSF-1 receptor), and CD11c were the best markers for distinguishing these cell types&#160;10.
Here, we present a method for isolation of cells at several distinct stages of development along the pathway of differentiation driven by GM-CSF: Common Myeloid Progenitor (CMP), Granulocyte-Macrophage Progenitor (GMP), monocyte, monocyte-derived Macrophage (MoMac) and monocyte-derived DC (MoDC). The moMac population can be further segregated based on level of MHC class II expression, revealing a moDC precursor population (moDP)&#160;10. We utilize a high-speed fluorescence-activated cell sorting (FACS) strategy to isolate these 5 populations based on expression of Ly6C, CD115, and CD11c. We then demonstrate the examination of these cells in functional assays revealing their responses to PAMP stimulation.

<table>
	<tbody>
		<tr>
			<td>RPMI 1640</td>
			<td>Corning</td>
			<td>15-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum (FCS)</td>
			<td>HyClone</td>
			<td>SV30014.04</td>
			<td>to supplement complete medium and FWB</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050</td>
			<td>to supplement complete medium</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol (2-ME)</td>
			<td>MP Biomedical</td>
			<td>190242</td>
			<td>to supplement complete medium</td>
		</tr>
		<tr>
			<td>75mM Vacuum Filter</td>
			<td>Thermo Scientific</td>
			<td>156-4045</td>
			<td>to sterilize complete media</td>
		</tr>
		<tr>
			<td>ACK Lysis Buffer</td>
			<td>Lonza</td>
			<td>10-548E</td>
			<td>to lyse red blood cell</td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Corning</td>
			<td>21-020-CM</td>
			<td>to rescue leukocytes after red blood cell lysis</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS), Dulbecco&#39;s</td>
			<td>Lonza</td>
			<td>17-512F</td>
			<td>must be endotoxin free; chilled at 4 &#176;C</td>
		</tr>
		<tr>
			<td>35&#181;m Cell filter</td>
			<td>Falcon</td>
			<td>352235</td>
			<td>to break apart clumps before running through cytometer.</td>
		</tr>
		<tr>
			<td>GM-CSF</td>
			<td>Biosource</td>
			<td>PMC2011</td>
			<td>usable concentration of 10ng/mL</td>
		</tr>
		<tr>
			<td>Tissue cultured treated plate</td>
			<td>VWR</td>
			<td>10062-896</td>
			<td>for bone marrow cells after harvest</td>
		</tr>
		<tr>
			<td>Anti-Ly6C, Clone HK1.4</td>
			<td>Biolegend</td>
			<td>128018</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD115, Clone AFS98</td>
			<td>Tonbo Bioscience</td>
			<td>20-1152-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD11c, Clone HL3</td>
			<td>BD Biosciences</td>
			<td>557400</td>
			<td>to differeniate CMP and MoDCs</td>
		</tr>
		<tr>
			<td>MoFlo XPD Flow Cytometer</td>
			<td>Beckman Coulter</td>
			<td>ML99030</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Accuri C6</td>
			<td>BD Biosciences</td>
			<td>660517</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Pharmco-Aaper</td>
			<td>111000200CSPP</td>
			<td></td>
		</tr>
		<tr>
			<td>60mm Petri Dish</td>
			<td>Corning, Inc</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Conical tube</td>
			<td>VWR</td>
			<td>21008-242</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 Mice</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td>Female; 10-20 weeks old</td>
		</tr>
		<tr>
			<td>Biosafety Hood</td>
			<td>Thermo Scientific</td>
			<td>8354-30-0011</td>
			<td></td>
		</tr>
		<tr>
			<td>10mL Syringe</td>
			<td>BD Biosciences</td>
			<td>301604</td>
			<td></td>
		</tr>
		<tr>
			<td>23-gauge needle</td>
			<td>BD Biosciences</td>
			<td>305145</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810&#160;R</td>
			<td>eppendorf</td>
			<td>22625501</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo Software v10</td>
			<td>BD Biosciences</td>
			<td>Version 10</td>
			<td>flowjo.com</td>
		</tr>
	</tbody>
</table>

generation of large numbers of myeloid progenitors and dendritic cell precursors from murine bone marrow using a novel cell sorting strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

In an effort to keep as many channels available for analysis as possible, viable cells were routinely selected based on forward and side scatter, excluding very small and very granular events (a typical gate is applied to all the dot plots in Figure 1A). To determine if this gating strategy reliably excluded dead cells, we stained with 7-Amino actinomycin D (7-AAD) (Figure 1B). 7AAD stains DNA in dead and dying cells due to membr...

Watch this Scientific Journal Video about Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy at JoVE.com

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

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