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 of a 0.22 &#181;m vacuum filter flask unit, and apply vacuum. 
	NOTE: Complete medium can be stored at 4 &#176;C for up to 2 months.</li>
	<li>Prepare 70% ethanol solution by mixing 350 mL of 100% ethanol with 150 mL sterile H2O in a 500 mL flask.</li>
	<li>Set centrifuge to 4 &#176;C.</li>
	<li>Sterilize forceps, scalpel, and scissors in 70% ethanol.</li>
	<li>Using a serological pipette, add 5 mL of complete media to three 60 mm Petri dishes.</li>
	<li>Using a serological pipette, add 30 mL of complete media to a 50 mL conical tube.</li>
</ol>
2. Collection of Murine Bone Marrow Cells
<ol>
	<li>Euthanize a C57BL/6 mouse by CO2 narcosis in accordance with the rules established by the 2013 American Veterinary Medical Association (AVMA) Guidelines on Euthanasia.</li>
	<li>Remove and strip hind legs.
	<ol>
		<li>Saturate the hind legs and torso with 75% ethanol, and make shallow cuts through the skin around the hip joint with curved tissue scissors. Using forceps, firmly pull the skin from the hip down towards the ankle, revealing the muscle. Use scissors to remove the skin flap.</li>
		<li>Remove the whole hind leg by cutting through the bone just above the femur/hip joint. 
		NOTE: In addition to sterilizing the area, the ethanol will aid in providing clean cuts and prevent hair from contaminating the samples.</li>
		<li>If the legs will be transferred to a new location for bone marrow harvesting, submerge the legs in complete media.</li>
	</ol>
	</li>
	<li>Working in a sterile biosafety cabinet, transfer the legs to one of the previously prepared Petri dishes.</li>
	<li>Use scissors to cut the just below the ankle, and carefully remove as much of the muscle and elastic connective tissue as possible. Transfer the cleaned bone to the second prepared Petri dish. 
	NOTE: Although it is not necessary to remove all the muscle, too much remaining tissue can make it difficult to flush out the bone marrow.</li>
	<li>Separate the femur, knee, and tibia.
	<ol>
		<li>Using forceps, hold the leg at the knee and locate the marrow. 
		NOTE: Bone marrow should be visible as a faint red line inside the bone cavity at the top of the femur and toward the end of the tibia.
		<ol>
			<li>Using scissors, make three cuts as follows.
			<ol>
				<li>Cut the tibia just above where the marrow appears to end.</li>
				<li>Cut just below the knee joint.</li>
				<li>Cut just above the knee joint.</li>
				<li>If the hip joint is still connected to the femur, cut just below the hip joint.</li>
			</ol>
			</li>
			<li>Return the three fragments to the Petri dish and repeat this process on other leg.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Flush bone marrow from the femur and tibia.
	<ol>
		<li>Fill a 10 mL syringe with complete media from the 50 mL conical tube, and cap with 23 G needle.</li>
		<li>Holding the bone with forceps above the third prepared Petri dish, insert the needle into the bone canal and push media through, flushing out the cells (which may emerge as an intact &#34;plug&#34; or as several clusters). Repeat until no more color can be seen through the bone. Refill the syringe with media as necessary.</li>
	</ol>
	</li>
	<li>Crush the epiphyses.
	<ol>
		<li>While still in the second Petri dish, hold the knee cap firmly with forceps, and mash the knees with the tip of the syringe. Continue until the epiphyses are no longer red.</li>
	</ol>
	</li>
	<li>Using the syringe, transfer the cells from the second and third Petri dish to the 50 mL tube. Breakup up clumps by gently pipetting up and down. Try not to generate bubbles. Centrifuge at 250 x g at 4 &#176;C for 10 min.</li>
	<li>Lyse red blood cells
	<ol>
		<li>Remove supernatant with serological pipette, dislodge pellet by flicking, and lyse red blood cells by incubating in 1 mL of ACK (Ammonium-Chloride-Potassium) Lysis Buffer for 1 min at room temperature.</li>
		<li>Using a serological pipette, add 40 mL of HBSS (Hanks Balanced Salt Solution) buffer.</li>
	</ol>
	</li>
	<li>Using a serological pipette, filter the cells though a 70 &#181;m cell strainer into a new 50 mL conical tube. Centrifuge at 250 x g at 4 &#176;C for 10 min.</li>
	<li>Using a serological pipette, remove supernatant and wash cells with 40 mL of complete media. Centrifuge at 250 x g at 4 &#176;C for 10 min. 
	NOTE: At this stage, lineage positive lymphocytes can be removed by FACS or magnetic column purification. However, lymphocytes are not maintained long term in culture. Although a large number of lineage positive cells are present in the Ly6C-CD115- population in the bone marrow ex vivo, nearly all are absent by day 5 (Figure 2).</li>
	<li>Using a serological pipette, remove the supernatant, and culture the bone marrow cells in complete media with 10 ng/mL of recombinant mouse GM-CSF at a density of 1 x 106 cells/mL. 
	NOTE: Typically, 4 x 107 total cells can be harvested after red blood cell lysis. However, expect as little as 2 x 107 for beginners and up to 5 x 107 cells for experienced harvesters.</li>
	<li>Using a serological pipette, transfer the cells to tissue culture plates, and incubate at 37 &#176;C in 5% CO2. If using a 24-well plate, seed each well with 2 mL of cell suspension.</li>
	<li>Every 48 h, use a serological pipette to remove half of the media and replace with fresh complete media and GM-CSF. 
	NOTE: Cultures can be kept up to 9 days. However, composition changes over time. See Section 3 for more information.</li>
</ol>
3. Choosing Day of Sort
<ol>
	<li>As cell population compositions change over time, select a day that yields the highest number of desired cells.</li>
	<li>See Table 1 for the expected cell yield post sort for each of the populations after 3, 5, and 7 days of culture in GM-CSF per 1 x 107 cells.</li>
</ol>
4. Staining strategy
<ol>
	<li>Use small aliquots of cells to prepare control samples. Include an unstained control, compensation control samples stained with only one fluorescent antibody each, and fluorescence-minus-one controls in which all antibodies are added except one, to control for non-specific fluorescence in that channel. If using indirect labeling, include primary alone, secondary alone, and both primary and secondary. 
	NOTE: Phycoerythrin (PE) and allophycocyanin (APC) tagged antibodies provide distinct separation with minimal bleed over when used together. However, if fluorochrome options are limited, CD115 is expressed at a relatively low level, while Ly6C is expressed at very high levels. Therefore, brighter fluorochromes are desirable for anti-CD115, and anti-Ly6C fluorochromes are chosen to prevent bleed over.</li>
	<li>Prepare 100 mL of FACS Wash Buffer (FWB) by mixing 97 mL of chilled Dulbecco&#39;s phosphate buffered saline (DPBS) with 3 mL of fetal calf serum in a 50 mL conical tube, and place in an ice bath.</li>
	<li>Use a pipette to gently, but thoroughly, pipette cells up and down to dislodge any loosely adherent cells.</li>
	<li>Using a serological pipette, transfer cells to a 50 mL conical tube. Centrifuge at 250 x g at 4 &#176;C for 10 min.
	<ol>
		<li>If cell volume exceeds 50 mL, transfer cells to the necessary number of tubes and combine at the staining step.</li>
	</ol>
	</li>
	<li>Gently pour off the supernatant, and wash the pelleted cells by adding 30 mL of FWB with a serological pipette. Centrifuge at 250 x g at 4 &#176;C for 10 min, and repeat the wash. 
	NOTE: If high cell death is expected, cells can be washed with FWB with as low as 0.5% fetal calf serum (FCS). This will prevent cell clumping.</li>
	<li>Suspend and stain cells per antibody manufacturer&#39;s instructions.
	<ol>
		<li>Suspend 5 x 107 cells in 1 mL of FWB and add 2 &#181;g each of anti-Ly6C and anti-CD115 labeled with the fluorophores (of the researcher&#39;s choice). To further distinguish CMP from moDC (both are Ly6C- and CD115-), add 2 &#181;g of anti-CD11c antibodies (CMP are CD11c-; moDC are CD11c+). Incubate for 30 min on ice. 
		NOTE: Figure 3 was generated using Ly6C-PE and CD115-APC.</li>
	</ol>
	</li>
	<li>Using a serological pipette, add 10 mL of FWB, and centrifuge at 250 x g at 4 &#176;C for 10 min.</li>
	<li>Gently pour off the supernatant, and wash the pelleted cells by adding 30 mL of FWB with a serological pipette. Centrifuge at 250 x g at 4 &#176;C for 10 min, and repeat the wash.</li>
	<li>Before suspending cells, flick tube thoroughly to dislodge the pellet. Use a serological pipette to suspend cells at 1 x 107 cell/mL of FWB, and filter through 35-&#181;m cell filter. Use a serological pipette to transfer filtered cells into polypropylene tube, and place on ice until ready to sort. 
	NOTE: If polypropylene is unavailable, protein coated (nonfat dry milk or FCS) polystyrene tubes can be used to reduce binding.</li>
</ol>
5. Set Gates Based on Control Samples
NOTE: To prevent cell disruption due to the pressure of the high-speed flow stream, use a 100&#8211;130 &#181;m nozzle for cell sorting.
<ol>
	<li>Run the unstained control (see step 4.1) through the cell sorter, and apply a gate to exclude small debris (low forward scatter; FSC) and highly granular (high side scatter; SSC) particles.
	<ol>
		<li>To analyze only the later stages (monocytes, moMac/MoDP, and MoDC), apply gating to only include larger cells (high FSC).</li>
		<li>If viability stains are being used, use these to exclude stained, non-viable events (an example is illustrated in Figure 1).</li>
	</ol>
	</li>
	<li>Run the single fluorescent control samples through the cell sorter, and adjust compensation as needed.</li>
	<li>Run a sample of the multi-labeled sample. Observe four distinct populations: Ly6C+CD115- (GMPs), Ly6C+CD115+ (monocytes), Ly6C-CD115+ (moMacs/moDP), and Ly6C-CD115- (CMPs/moDCs). Apply a gate to isolate each of the four major populations. 
	NOTE: CMPs and moDC share the Ly6C-CD115- phenotype. However, they can be differentiated based on CD11c expression: CMP lack CD11c, whereas MoDCs express CD11c.</li>
</ol>
6. Collection of Isolated Populations
<ol>
	<li>Prepare collection tubes by adding enough FCS to achieve at least 20% final concentration when full. For example, if using 5 mL tubes, add 1 mL of FCS before sorting, and remove the tube when it reaches 5 mL total volume.</li>
	<li>To prevent membrane turnover and antibody uptake, keep all samples (mixed and sorted) at 4 &#176;C throughout the sort.
	<ol>
		<li>If this is not possible, keep the tube containing the cells to be sorted on ice as much as possible and transfer aliquots to the sorter as needed.</li>
		<li>Additionally, transfer sorted samples to ice every 20&#8211;30 min.</li>
	</ol>
	</li>
	<li>After the desired number of cells have been collected, use a serological pipette to transfer the cells to a new conical tube. Centrifuge at 250 x g at 4 &#176;C for 10 min.
	<ol>
		<li>Confirm purity with post-sort analysis on small aliquots from each collected population.</li>
	</ol>
	</li>
	<li>Remove the supernatant, suspend in 10 mL of FWB and centrifuge at 250 x g at 4 &#176;C for 10 min. Repeat for a total of two washes. 
	NOTE: It can be difficult to remove all the supernatant without dislodging the pellet. Attaching a pipette tip to a vacuum line can help with removal. If this is not available, it is suggested that the supernatant be collected in a fresh tube in case of pellet dissociation.</li>
	<li>Remove the supernatant after the second wash.</li>
	<li>If the user&#39;s experimental design dictates the cells be re-cultured, follow steps 2.12&#8211;2.14. Otherwise, if cells will be used for immediate analysis, prepare cells according to desired protocol. 
	NOTE: An example of typical functional analysis is included in Figure 4.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+dendritic+cell+development+by+GM-CSF:+molecular+control+and+implications+for+immune+homeostasis+and+therapy.">Regulation of dendritic cell development by GM-CSF: molecular control and implications for immune homeostasis and therapy.</a> Blood. 119, 3383-3393 (2012).</li><li>Steinman, R. M., Inaba, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+dendritic+cells.">Myeloid dendritic cells.</a> J Leukoc Biol. 66, 205-208 (1999).</li><li>Inaba, 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=Generation+of+large+numbers+of+dendritic+cells+from+mouse+bone+marrow+cultures+supplemented+with+granulocyte/macrophage+colony-stimulating+factor.">Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.</a> J Exp Med. 176, 1693-1702 (1992).</li><li>Steinman, R. M., Pack, M., Inaba, K. <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+development+and+maturation.">Dendritic cell development and maturation.</a> Adv Exp Med Biol. , 1-6 (1997).</li><li>Pierre, 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=Developmental+regulation+of+MHC+class+II+transport+in+mouse+dendritic+cells.">Developmental regulation of MHC class II transport in mouse dendritic cells.</a> Nature. 388, 787-792 (1997).</li><li>Steinman, R. M., Witmer-Pack, M., Inaba, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells:+antigen+presentation,+accessory+function+and+clinical+relevance.">Dendritic cells: antigen presentation, accessory function and clinical relevance.</a> Adv Exp Med Biol. , 1-9 (1993).</li><li>Na, Y. R., Jung, D., Gu, G. J., Seok, 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=GM-CSF+Grown+Bone+Marrow+Derived+Cells+Are+Composed+of+Phenotypically+Different+Dendritic+Cells+and+Macrophages.">GM-CSF Grown Bone Marrow Derived Cells Are Composed of Phenotypically Different Dendritic Cells and Macrophages.</a> Mol Cells. 39, 734-741 (2016).</li><li>Rogers, P. B., Driessnack, M. 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. 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The authors have no conflicts to disclose.

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 prior studies.
Previous protocols for isolation of progenitors and precursors generated from bone marrow in vitro have relied on CFSE-staining to identify proliferative progenitor cells&#160;17 or on staining with CD31 and Ly6C as markers of myeloid cells&#160;18. The CFSE staining protocol described by Naik was designed for use in generation of Flt3L-driven DC progenitors and precursors&#160;17. When we attempted this approach in the GM-CSF culture system, we encountered two main issues. The CFSE was somewhat cytotoxic to the developing cells and was so bright (even in divided cells) that it made compensation difficult. This approach proved to be unsuitable for our goals of isolating early cell types (progenitors), as well as cells across the developmental spectrum, from highly proliferative (CMPs/GMPs) to highly developed (MoDC/MoMac). We also tried the sorting strategy for GM-CSF-driven cells described by Leenen&#39;s group which was based on CD31 and Ly6C&#160;18. However, we found that CD31 was problematic. It was expressed at very low levels (making it difficult to resolve the populations for clean sorting) by a vanishingly small subset of cells, and only very early during the culture period. In fact, CD31 expression was not detectable past day 2 of culture in GM-CSF&#160;10.
Ly6C, however, was a very useful molecule for separating cells at different stages of development due to its transient pattern of expression&#160;10. The addition of CD115 enabled us to more closely discern cells at intermediate and later stages of development that was not possible with CD31. We also examined other markers of progenitors such as CD34 and CD117 in hopes of isolating large numbers of these early cells&#160;10. However, unlike that reported in the Flt3L system, we found that CD34 and CD117 were also expressed on a very small subset of cells and were virtually absent by day 3 of culture&#160;17. These stem cell markers may be useful in future studies however, to distinguish subsets within the CMP or GMP populations.
There are several potential modifications to the protocol that may affect the yield of desired cell populations. First, the user may choose to apply a viability stain to exclude any dead cells. Based on the observations, the rate of dead cells within a typical forward and side scatter gate are relatively low in general, and pose problems only during the first few days of culture, coincident with decreases in lineage positive lymphocytes. However, for applications in which cell numbers must be precise, a viability stain will ensure exclusion of dead cells.
A second potential modification is depletion of lineage-positive lymphocytes prior to culture. We have tried this approach to address the small population of lineage positive cells that were regularly observed within the Ly6C-CD115- cell population. The overall cell yield was slightly lower at days 5 and 6, and the purity was not significantly higher (data not shown). Thus, the purity did not justify the reduced yield. Likewise, if the user plans to sort on day 5 or after, the frequency of lymphocytes within the culture is well below 1% and should not present a problem.
Finally, because this strategy is designed to isolate cells across a large developmental spectrum the user should tailor the timing of their sort to collect as many of the desired populations as possible. This sorting strategy faithfully yields the cell types indicated regardless of the day of culture on which the cells are sorted. For example, cells with the phenotype Ly6C+ CD115- CD11c- are true to the GMP phenotype and function similarly whether they are sorted and isolated on day 3 or 5 of culture. However, the frequency of these cells is much greater on day 3 than 5, so if the goal is isolation of this cell type, sorting on day 3 would be recommended. It is also notable that cells sorted on day 3 progress through the subsequent developmental stages with slower kinetics than if they were sorted on day 5 or 7 (Figure 3).
While this method allows for the clear delineation and isolation of 5&#8211;6 distinct populations along the developmental spectrum, there are likely many more populations or transitional stages along this pathway. Within each of the five described populations there may be several sub-populations at slightly different increments of development. For example, we have observed &#34;distinct&#34; populations with intermediate levels of CD115 and of Ly6C which undergo slightly different patterns of development, in terms of how many moMac and moDC are generated (data not shown). It is also likely that populations previously observed in vivo are present in the culture and sorting system, but these have been difficult to identify due to their very low frequency. Populations such as cMOP&#160;19, MDP&#160;20, or CDP&#160;21 are likely present, but may be obscured within the larger populations. Future studies will be needed to further clarify the numerous developmental stages that may be isolated within this sorting framework with the addition of specific markers of differentiation. The value of this sorting strategy is that it allows consistent isolation of large numbers of developmentally distinct stages during DC ontogeny.

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>75 mM 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>HBSS 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&#039;s</td><td>Lonza</td><td>17-512F</td><td>must be endotoxin free; chilled at 4 &amp;deg;C</td></tr><tr><td>35 &amp;micro;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 10 ng/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>60 mm Petri Dish</td><td>Corning, Inc</td><td>353002</td><td></td></tr><tr><td>50 mL 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>10 mL Syringe</td><td>BD Biosciences</td><td>301604</td><td></td></tr><tr><td>23 G needle</td><td>BD Biosciences</td><td>305145</td><td></td></tr><tr><td>Centrifuge 5810&amp;nbsp;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 membrane permeability, and is excluded by viable cells. Viability of GM-CSF cultured bone marrow cells was analyzed immediately post-harvest (PH) and 1, 3, and 5 PH. Cell analyzed immediately PH had approximately 10% of 7-AAD positive cells when a typical FSC/SSC gate was applied to freshly isolated cells from the bone marrow (Figure 1, Post-Harvest). A similar proportion of dead cells was also present at day 1 (~12%) and day 3 (~11%) of culture (Figure 1, 1 day PH and 3 days PH). By day 5, the number of dead cells within the gate was reduced to ~5% (Figure 1, 5 days PH). Thus, using such a viability gate is generally appropriate for sorting on day 5 and after. Therefore, if users are limited in their available parameters, FSC/SSC gating is generally appropriate. However, for more sensitive assays (particularly if they rely on precise cell numbers), incorporate a viability stain (suggestion).
Many protocols for propagation of dendritic cells recommend depletion of lineage positive cells, especially lymphocytes, from bone marrow prior to culture&#160;11,17. This procedure is thought to increase the purity of the cells recovered upon GM-CSF-mediated differentiation. Typically, cells expressing markers of T cells (CD3), B cells (CD45R or CD19), and NK cells (NK1.1) may be depleted by positive selection using magnetic beads or cell sorting&#160;11,17,18. However, based on the culturing system, lymphocytes were rarely recovered or detected in these assays. Thus, we sought to assess the longevity of lymphocytes maintained in the Ly6C-CD115- population among the GM-CSF-driven cells (Figure 2A). GM-CSF cultured bone marrow cells (that had not been lineage depleted) were stained with antibodies to CD3, CD45R, and NK1.1 (in the same fluorophore) and measured daily by flow cytometry (Figure 2B). Within the Ly6C-CD115- population, CD3/CD45R positive cells persisted strongly through Days 0&#8211;3 (Figure 2B). On Day 4, only a few CD3/CD45R positive cells remained, and by Day 5 and 6, there were no CD3/CD45R expressing cells present. Thus, within 4 days of culture in GM-CSF, lineage positive cells were essentially absent and were not detected at all at days 5 and 6 of culture.
The composition of the GM-CSF-driven cell culture changes daily in this system as the cells develop and differentiate (Table 1; Figure 3). At early time points, the most abundant cells are progenitors and precursors, and at later times the majority of cells are more differentiated&#160;10. To determine how sorting on different days of culture might affect the subsequent developmental path or kinetics, sorts were performed 3, 5, and 7 days PH (Figure 3A). The development of the MoMac population (Ly6C-CD115+) was then tracked over 2&#8211;3 further days in culture (Figure 3B-3D).
When sorted 3 days PH, only 40% of Ly6C-CD115+ cells had decreased CD115 expression within 24 h post-sort (PS) (Figure 3B). By 48 h PS, the fraction that had down-regulated CD115 was 66%, and by 72 h, 70% of the cells had this phenotype. This phenotypic composition was maintained (~70-72% Ly6C-CD115-) even after further days of culture (data not shown). When sorted 5 days PH, ~75% of the cells were Ly6C-CD115-, having rapidly down-regulated CD115 within 24 h PS, and ~80% were CD115- after only 48 h. This distribution was maintained after 72 h (Figure 3C). Finally, when sorted 7 days PH, down-regulation of CD115 was also quite rapid. Within 24 h PS, ~75% of cells had down-regulated CD115 expression, and this trend was maintained after 48 h (Figure 3D). Interestingly, when sorted at day 7, the overall level of CD115 expression was lower on the cells within the CD115+ population.
Thus, these findings indicate that the kinetics of development are somewhat slower when sorting at an early day, such as day 3 sorted cells compared to the more rapid development and differentiation observed after sorting on day 5 or 7. Based on these results, a user seeking larger numbers of moDC should likely sort on day 5 or 7.
The maturation response of DC is well established 6,7,12,14. When treated with a variety of pathogen-associated molecular patterns (PAMPs), immature DC up-regulate the expression of MHC, costimulatory molecules, and pro-inflammatory cytokines, enhancing their T cell-activating capacity&#160;6. However, it is less clear when developing cells gain the capacity to respond to PAMP stimulation and which feature of DC maturation they might exhibit. To determine which of the sorted populations would respond to maturation stimuli, each population was treated shortly after sorting with a cocktail of PAMPs including Poly I:C, lipopolysaccharides (LPS), and CpG DNA to trigger toll-like receptor (TLR) 3 (TLR3), TLR4, and TLR9, respectively. Cells were treated (or untreated) for 24 h and expression of CD86 and MHCII was measured by flow cytometry (Figure 4A-4B). Furthermore, IL-12p40/70 and IL-6 production was measured in the supernatants by cytokine array ELISA (Figure 4C-4D).
CMPs and GMPs expressed very low levels of MHC class II and CD86 in an unstimulated state, and these expression patterns did not change significantly upon exposure to the cocktail of PAMPs (Figure 4A and 4B). Likewise, the expression of MHC class II by the monocytes was also low and changed little upon PAMP exposure (Figure 4A). However, the expression of CD86 was moderate on the monocytes 24 h after sorting, and it increased further following 24 h stimulation. Higher basal expression of MHC class II and CD86 in the MoMacs and MoDCs was observed, and both populations exhibited a strong induction of these molecules upon PAMP stimulation. In terms of MHC class II expression following stimulation, there was no statistical difference between the CMPs, GMPs, and monocytes, while the MoMacs and MoDCs formed a distinct group. Yet, in terms of CD86 expression, the CMPs and GMPs cells were statistically different than MoMacs and MoDCs. However, monocytes were not different than MoMacs or MoDCs.
We next wanted to assess the relative ability of each of the five populations to produce cytokines in response to TLR stimulation. Thus, we performed a cytokine assay on the sorted populations after culture in the presence or absence of the TLR agonist cocktail used above. Cells were cultured with the stimuli for 24 h, then supernatants were collected. We observed very little IL-12p40/70 or IL-6 production by CMPs upon TLR stimulation (Figure 4C-4D). The second population, GMPs, were unable to produce IL-12p40/70 (Figure 4C), but these cells produced a low amount of IL-6 upon stimulation by PAMPs (Figure 4D). The highest levels of cytokine production were observed in the latter three populations. Monocytes and MoMacs had very similar patterns and magnitudes of cytokine production. Both greatly increased IL-12p40/70 and modestly increased IL-6 production up stimulation (Figure 4C-4D). Interestingly, in the presence of PAMPs MoDCs did not increase IL-12p40/70 secretion; however, this population greatly increased IL-6 production (Figure 4C-4D). These findings indicate that the sorted populations maintained their immunological functions after isolation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57365/57365fig1.jpg" /> 
Figure 1: Viable cells within Forward versus Side Scatter gate. Mouse bone marrow was cultured in GM-CSF, and viability was measured using 7-AAD (7-amino-actinomycin D) staining post-harvest (PH) and 1, 3, and 5 days post-harvest. (A) Viable cells were selected by applying a gate (Viable) which omitted small and highly granular events based on FSC and SSC. (B) Histograms of 7-AAD staining generated from events within Viable (FSC/SSC) gate. Events positive for 7-AAD indicate cells undergoing apoptosis. Arrows indicate viable cell gating. <a href="/files/ftp_upload/57365/57365fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57365/57365fig2.jpg" /> 
Figure 2: Lymphocytes within the Ly6C-CD115- population. Mouse bone marrow was cultured in GM-CSF and lymphocyte markers (CD3, CD145R, and NK1.1) were analyzed daily by flow cytometry. (A) Cell were stained with Ly6C-PE and CD115-APC to identify Ly6C-CD115- cells. Quadrant gate was applied based on single-color controls. Pseudocolor dot plot generated from day 0 (B) CD3, CD45R, and NK1.1 expression of Ly6C-CD115- cells was analyzed on day of harvest (day 0) until day 6. Cell counts were normalized to mode using a flow cytometry analysis software. Arrow indicates Ly6C-CD115- gating. <a href="/files/ftp_upload/57365/57365fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57365/57365fig3.jpg" /> 
Figure 3: Kinetics of development of Ly6C-CD115+ cells after sorting on different days. (A) Mouse bone marrow was harvested and cultured in GM-CSF. Aliquots of 1 x 107 cells were harvested (B) 3, (C) 5, or (D) 7 days post-harvest (PH). On the indicated days PH, Ly6C-CD115+ cells were sorted from mixed culture (Pre-sort) and analyzed immediately post-sort (PS). Sorted cells were re-cultured in GM-CSF, and changes in Ly6C/CD115 expression were analyzed daily by flow cytometry. Box and arrow indicate sorting gate. <a href="/files/ftp_upload/57365/57365fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57365/57365fig4.jpg" /> 
Figure 4: Maturation and cytokine expression following TLR stimulation. Cells were sorted into 5 populations on day 3 of culture in GM-CSF. They were then treated with a cocktail of PAMPs (LPS, Poly I:C, and CpG DNA) for 24 h. Mean fluorescent intensity (MFI) of (A) MHC class II and (B) CD86 was analyzed immediately post-sort (PS) and 24 h with and without PAMPs by flow cytometry. Error bars represent standard deviation of 3-5 replicate experiments. (C) IL-12p40/70 and (D) IL-6 in supernatants from 3 pooled samples were measured by cytokine array dot blot ELISA after 24 h with and without PAMPs. RLU; Relative light units; -/+ indicate the presence of the cell surface marker for the designated population. <a href="/files/ftp_upload/57365/57365fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2" style="text-align: center;">Day 3</td>
			<td colspan="2" style="text-align: center;">Day 5</td>
			<td colspan="2" style="text-align: center;">Day 7</td>
		</tr>
		<tr>
			<td>Phenotype</td>
			<td style="text-align: left;">Cell type</td>
			<td style="text-align: center;">Min</td>
			<td style="text-align: center;">Max</td>
			<td style="text-align: center;">Min</td>
			<td style="text-align: center;">Max</td>
			<td style="text-align: center;">Min</td>
			<td style="text-align: center;">Max</td>
		</tr>
		<tr>
			<td style="text-align: left;">Ly6C-CD115-CD11c-</td>
			<td style="text-align: left;">CMP</td>
			<td style="text-align: center;">3 x 106</td>
			<td style="text-align: center;">4 x 106</td>
			<td style="text-align: center;">5 x 105</td>
			<td style="text-align: center;">1 x 106</td>
			<td style="text-align: center;">N/a</td>
			<td style="text-align: center;">N/a</td>
		</tr>
		<tr>
			<td style="text-align: left;">Ly6C+CD115-</td>
			<td style="text-align: left;">GMP</td>
			<td style="text-align: center;">2 x 106</td>
			<td style="text-align: center;">3 x 106</td>
			<td style="text-align: center;">2 x 106</td>
			<td style="text-align: center;">3 x 106</td>
			<td style="text-align: center;">N/a</td>
			<td style="text-align: center;">N/a</td>
		</tr>
		<tr>
			<td style="text-align: left;">Ly6C+CD115+</td>
			<td style="text-align: left;">Mono</td>
			<td style="text-align: center;">2 x 106</td>
			<td style="text-align: center;">3 x 106</td>
			<td style="text-align: center;">2 x 106</td>
			<td style="text-align: center;">3 x 106</td>
			<td style="text-align: center;">5 x 105</td>
			<td style="text-align: center;">1 x 106</td>
		</tr>
		<tr>
			<td style="text-align: left;">Ly6C-CD115+</td>
			<td style="text-align: left;">MoMac</td>
			<td style="text-align: center;">5 x 105</td>
			<td style="text-align: center;">1 x 106</td>
			<td style="text-align: center;">3 x 106</td>
			<td style="text-align: center;">4 x 106</td>
			<td style="text-align: center;">3 x 106</td>
			<td style="text-align: center;">4 x 106</td>
		</tr>
		<tr>
			<td style="text-align: left;">Ly6C-CD115-CD11c+</td>
			<td style="text-align: left;">MoDC</td>
			<td style="text-align: center;">N/a</td>
			<td style="text-align: center;">N/a</td>
			<td style="text-align: center;">5 x 105</td>
			<td style="text-align: center;">1 x 106</td>
			<td style="text-align: center;">3 x 106</td>
			<td style="text-align: center;">4 x 106</td>
		</tr>
	</tbody>
</table>
Table 1: Expected minimum and maximum number of cells recovered post-sort per 1 x 107 cells. CMP (common myeloid progenitor); GMP (granulocyte/macrophage progenitor) Mono (monocytes); MoMac (monocyte-derived macrophage); MoDC (monocyte-derived dendritic cell); N/a (not available); Min (minimum cell yield out of 1 x 107 cells); Max (maximum cell yield out of 1 x 107 cells).

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 ...

generation-large-numbers-myeloid-progenitors-dendritic-cell

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

9.0K Views.  Auburn University. 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.

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

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Biology

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers. Here we present a detailed description, readout, and interpretation of proliferation and differentiation assays which are used for the investigation of processes involved in myelopoiesis and neutrophilic differentiation. These experiments employ the 32D/G-CSF-R cytokine dependent murine myeloid cell line, which possesses the ability to proliferate in the presence of IL-3 and differentiate in G-CSF. We provide optimized protocols for handling 32D/G-CSF-R cells and discuss major pitfalls and drawbacks that might compromise the described assays and expected results. Additionally, this article contains protocols for lentiviral and retroviral production, titration, and transduction of 32D/G-CSF-R cells. We demonstrate that genetic manipulation of these cells can be employed to successfully perform functional and molecular studies, which can complement results obtained with primary hematopoietic stem and progenitor cells or in vivo models.

Here detailed protocols for culturing the murine myeloid precursor 32D/G-CSF-R cell line, performing viral infections, and carrying out proliferation and differentiation assays are presented. This cell line is suitable for studying myeloid cell development, and the role of genes of interest in myeloid cell growth and neutrophilic differentiation.

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of ...

Developmental Biology

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming &#34;units&#34; known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Here, we describe a novel flow cytometric method for prospective isolation of early burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors directly from fresh mouse bone marrow and spleen. This protocol, developed based on single-cell transcriptomic data, is the first to isolate all the tissue's erythroid progenitors with high purity.

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...

Isolation of Mouse Megakaryocyte Progenitors

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through megakaryopoiesis. The final stages of this process are complex and classically involve the bipotent Megakaryocyte-Erythrocyte Progenitors (MEP) and the unipotent Megakaryocyte Progenitors (MKp). These populations precede the formation of bona fide megakaryocytes and, as such, their isolation and characterization could allow for the robust and unbiased analysis of megakaryocyte formation. This protocol presents in detail the procedure to collect hematopoietic cells from mouse bone marrow, the enrichment of hematopoietic progenitors through magnetic depletion and finally a cell sorting strategy that yield highly purified MEP and MKp populations. First, bone marrow cells are collected from the femur, the tibia, and also the iliac crest, a bone that contains a high number of hematopoietic progenitors. The use of iliac crest bones drastically increases the total cell number obtained per mouse and thus contributes to a more ethical use of animals. A magnetic lineage depletion was optimized using 450 nm magnetic beads allowing a very efficient cell sorting by flow cytometry. Finally, the protocol presents the labeling and gating strategy for the sorting of the two highly purified megakaryocyte progenitor populations: MEP (Lin-Sca-1-c-Kit+CD16/32-CD150+CD9dim) and MKp (Lin- Sca-1-c-Kit+CD16/32-CD150+CD9bright). This technique is easy to implement and provides enough cellular material to perform i) molecular characterization for a deeper knowledge of their identity and biology, ii) in vitro differentiation assays, that will provide a better understanding of the mechanisms of maturation of megakaryocytes, or iii) in vitro models of interaction with their microenvironment.

This method describes the purification by flow cytometry of MEP and MKp from mice femurs, tibias, and pelvic bones.

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through ...