This protocol was developed using funds from the Board of Governors Regenerative Medicine Institute at Cedars-Sinai Medical Center (to HSG), a Careers in Immunology fellowship from the American Association of Immunologists (to AY and HSG), and a Scholar Award from the American Society of Hematology (to AY). We thank the Flow Cytometry Core at Cedars-Sinai Medical Center for assistance with FACS sorting.

All methods described here were approved by the Institutional Animal Care and Use Committee (IACUC) of Cedars-Sinai Medical Center.
1. Isolation of Mouse Bone Marrow and Preparation of a Single Cell Suspension
<ol>
	<li>Euthanize the mouse in accordance with institutional guidelines.</li>
	<li>Place the euthanized mouse on its back and spray it with 70% ethanol (EtOH). Make a small (3 - 5 mm) incision in each hind limb at the ankle level using sharp, pointed scissors, and pull the skin up towards the body to remove it from the legs and expose the muscles and bones.</li>
	<li>Remove the muscles (quadriceps, hamstrings, etc.) from the bones by trimming them with scissors, following the direction of the bone. Be careful not to damage the bones.</li>
	<li>Dislocate the femurs from the hip joints and cut away the connective tissue and muscle to detach the legs, being careful not to cut into the bones.</li>
	<li>Remove the feet by dislocating the ankles from the tibias, place the bones in a tube of sterile PBS or media, and transport them on ice to the tissue culture room. 
	NOTE: For some other applications (e.g., deriving macrophages from total bone marrow cells) it is possible to leave the bones on ice for a while, but for progenitor isolation it is important to proceed as quickly as possible to avoid downregulation of important surface markers (especially CD115).</li>
	<li>In a biosafety cabinet, remove any remaining soft tissue from the bones by rubbing them with tissue paper. 
	Note: The protocol should be performed in a biosafety cabinet if a sterile sample is required for cell culture or in vivo assays after FACS sorting. If sterile cells are not required, the whole process can be performed on the lab bench.</li>
	<li>Briefly immerse the bones in 70% EtOH to disinfect them, and then in sterile PBS to wash them.</li>
	<li>Separate the femurs from the tibias and fibulas by cutting through the knee, and discard the fibulas.</li>
	<li>Cut the ends off of the bones with sharp scissors.</li>
	<li>Harvest bone marrow with cold sterile PBS as follows until the bones appear white:
	<ol>
		<li>Grip each bone with forceps, and insert a 26G needle connected to a 10 mL syringe containing cold sterile PBS into the bone shaft at one end.</li>
		<li>Hold the bone above a 50 mL centrifuge tube and flush 2 - 5 mL PBS through the bone to harvest the marrow.</li>
		<li>Harvest the marrow from all 4 bones per mouse (2 femurs and 2 tibias).</li>
	</ol>
	</li>
	<li>Gently pipette up and down with a 1 mL pipette to disaggregate the bone marrow to form a cell suspension.</li>
	<li>Filter the bone marrow suspension through a piece of 30 &#956;m nylon mesh to eliminate bone pieces and cell aggregates, thereby generating a single cell suspension.</li>
	<li>Centrifuge the single cell suspension at 280 x g for 5 min, and resuspend the cell pellet in sterile staining buffer (PBS + 0.5% FBS + 2 mM EDTA), using 5 mL staining buffer per mouse (e.g., 10 mL for cells pooled from the femurs and tibias of 2 mice).</li>
	<li>Take a 10 &#956;L aliquot of the sample, mix it with 10 &#956;L Trypan Blue, and load 10 &#956;L of the resulting Trypan Blue-labeled sample on to a hemocytometer. Count the cells using a light microscope. 
	NOTE: If the cells are too concentrated for reliable counting, dilute the sample prior to adding Trypan Blue.</li>
</ol>
2. Progenitor Enrichment by Magnetic-Activated Cell Sorting (MACS)
<ol>
	<li>Centrifuge the entire single cell suspension (or as many cells as are required to obtain the desired yield) at 280 x g for 5 min at 4&#176;C and resuspend the cell pellet in 40 &#956;L staining buffer (PBS + 0.5% FBS + 2 mM EDTA) per 107 cells. 
	NOTE: It is also possible to use MACS buffer from the lineage depletion kit manufacturer (see Table of Materials) as an alternative to the staining buffer.</li>
	<li>Perform the lineage depletion according to the instructions supplied with the lineage depletion kit. The following instructions are for the kit seen in the Table of Materials. If a different kit is used, follow the manufacturer&#39;s instructions and then proceed to step 2.3.
	<ol>
		<li>Add 10 &#956;L biotin-antibody cocktail per 107 cells, mix by pipetting and incubate for 10 min at 4 &#176;C.</li>
		<li>Add 30 &#956;L staining buffer/MACS buffer per 107 cells and mix.</li>
		<li>Add 20 &#956;L anti-biotin microbeads per 107 cells. Mix well and incubate for an additional 15 min at 4 &#176;C. 
		NOTE:&#160;Vortex the microbeads before adding them to the cells to make sure that they are completely dispersed.</li>
		<li>Add 1 mL staining buffer/MACS buffer per 107 cells, centrifuge the cells at 280 x g for 5 min, and resuspend the cell pellet in 500 &#956;L staining buffer/MACS buffer for up to 108 cells. For more than 108 cells, scale up the volume of staining buffer/MACS buffer accordingly.</li>
		<li>Prepare the automated magnetic separator according to the manufacturer&#39;s instructions.</li>
		<li>Place the tube containing the labeled cells into the separator and choose the negative selection program.</li>
		<li>Collect the negative fraction, which contains the progenitor-enriched lineage-negative (Lin-) cells. Discard the positive fraction, which contains the magnetically-labeled lineage-positive cells.</li>
	</ol>
	</li>
	<li>Centrifuge the Lin- cells at 280 x g for 5 min, and resuspend the cell pellet in 2 mL staining buffer/MACS buffer per mouse (e.g., 4 mL if bone marrow was pooled from 2 mice). 
	Note: The pellet should now be white instead of red because the erythrocytes have been depleted.</li>
	<li>Take a 10 &#956;L aliquot of the Lin- cells, mix it with 10 &#956;L Trypan Blue, and load 10 &#956;L of the resulting Trypan Blue-labeled sample on to a hemocytometer. Count the cells using a light microscope. 
	NOTE: If the cells are too concentrated for reliable counting, dilute the sample prior to adding Trypan Blue.</li>
</ol>
3. Myeloid Progenitor Identification and Isolation by Fluorescence-activated Cell Sorting (FACS)
<ol>
	<li>Label 9 microcentrifuge (1.5 or 2 mL) tubes for: 1) unstained cells, 2-8) cells single stained with fluorophore-conjugated antibodies against Fc&#947;R (CD16/32), c-Kit, Sca-1, CD34, Flt3 (CD135), Ly6C and CD115 for voltage selection and color compensation, and 9) sample cells to be stained with all 7 antibodies for progenitor identification and sorting.</li>
	<li>Distribute 100,000 cells to each of the control tubes (tubes 1 - 8), and all the remaining cells (or fewer if desired) to the sample tube (tube 9). 
	NOTE: If the sample volume is greater than 1.5 - 2 mL, it may be necessary to distribute the sample cells into a 15 mL tube, centrifuge them and resuspend the pellet (step 3.3 below), and then transfer the cells to a microcentrifuge tube for the subsequent staining steps.</li>
	<li>Centrifuge the cells at 1,500&#160;x g for 5 min, and resuspend the control tube pellets in 100 &#956;L staining buffer (PBS + 0.5% FBS + 2 mM EDTA) and the sample tube pellet in 100 &#956;L staining buffer per 5 x 106 cells (e.g., 200 &#956;L for 1 x 107 cells).</li>
	<li>Add 2 &#956;g/mL anti-CD16/CD32-APC-Cy7 to the Fc&#947;R single stain tube and the sample tube. Vortex gently to mix, and incubate for 10 min at 4 &#176;C. 
	NOTE: It is important to stain the sample cells with the Fc&#947;R (CD16/32) antibody prior to adding the other antibodies to prevent competition due to non-specific Fc-mediated antibody binding. Do not incubate the cells with an Fc&#947;R blocking reagent.</li>
	<li>Add the other antibodies to the corresponding single stain tubes (1 antibody each) and the sample tube (all 6 antibodies): 10 &#956;g/mL c-Kit-Pacific Blue, 2 &#956;g/mL Sca-1-PE-Cy7, 25 &#956;g/mL CD34-FITC, 10 &#956;g/mL Flt3-APC, 2 &#956;g/mL Ly6C-PerCP-Cy5.5, and 4 &#956;g/mL CD115-PE. Vortex gently, and incubate for 15 min at 4 &#176;C.</li>
	<li>Add 900 &#956;L staining buffer to the control tubes, and 1 mL staining buffer per 107 cells to the sample tube. Centrifuge the cells at 1,500 x g for 5 min and resuspend the cell pellet in staining buffer: 200 &#956;L per control tube, and 500 &#956;L per 25 x 106 cells in the sample tube. Transfer the resuspended cells to 5 mL FACS tubes. 
	NOTE: If the sample volume is greater than 1.5 - 2 mL, it may be necessary to transfer the cells to a 15 mL tube for centrifugation.</li>
	<li>Prepare the flow cytometer (analyzer or sorter) according to the manufacturer&#39;s instructions.</li>
	<li>Run the unstained and single stained control cells through the flow cytometer to set up the voltages and color compensations.</li>
	<li>Run the sample cells, gate the progenitor subsets as outlined below and summarized in Figure 2, and if desired, perform FACS sorting to isolate the relevant progenitor subsets. 
	NOTE: It is typically necessary to acquire approximately 200,000 cells to obtain at least 1,000 events in each progenitor gate.
	<ol>
		<li>Create a dot plot of all cells, displaying FSC-A (x-axis) and SSC-A (y-axis), and gate to exclude debris and dead cells =&#62; &#34;live&#34; cells.</li>
		<li>Create a dot plot of &#34;live&#34; cells, displaying FSC-H (x-axis) and FSC-W (y-axis), and gate to exclude doublets =&#62; live/singlet (FSC) gate.</li>
		<li>Create a dot plot of live/singlet (FSC) cells, displaying SSC-H (x-axis) and SSC-W (y-axis), and gate to exclude doublets =&#62; live/singlet (FSC)/singlet (SSC) gate.</li>
		<li>Create a dot plot of live/singlet (FSC)/singlet (SSC) cells, displaying Sca-1 (x-axis) and c-Kit (y-axis), and gate to select c-Kit+ Sca-1- cells =&#62; LKS- cells.</li>
		<li>Create a dot plot of LKS- cells, displaying CD34 (x-axis) and Fc&#947;R (y-axis), and gate to select CD34+ Fc&#947;Rlo cells =&#62; &#34;CMPs&#34;, and CD34+ Fc&#947;Rhi cells =&#62; &#34;GMPs&#34;. 
		Note: It&#8217;s important to gate the &#8220;CMPs&#8221; and &#8220;GMPs&#8221; as precisely as possible. It&#8217;s helpful to use a pseudocolor density plot or a contour plot for accurate gating.</li>
		<li>Create a dot plot of &#34;CMPs&#34;, displaying CD115 (x-axis) and Flt3 (y-axis), and gate to select: 
		Flt3+ CD115lo cells =&#62; CMP-Flt3+ CD115lo cells, 
		Flt3- CD115lo cells =&#62; CMP-Flt3- cells, 
		Flt3+ CD115hi cells =&#62; MDPs.</li>
		<li>Create a dot plot of &#34;GMPs&#34;, displaying Ly6C (x-axis) and Fc&#947;R (y-axis), and gate to select Ly6C-cells =&#62; &#34;GMP-Ly6C- cells&#34;, and Ly6C+ cells =&#62; &#34;GMP-Ly6C+ cells&#34;.</li>
		<li>Create a dot plot of &#34;GMP-Ly6C- cells&#34;, displaying CD115 (x-axis) and Flt3 (y-axis), and gate to select: 
		Flt3- CD115lo cells =&#62; GMPs.</li>
		<li>Create a dot plot of &#34;GMP-Ly6C+ cells&#34;, displaying CD115 (x-axis) and Flt3 (y-axis), and gate to select: 
		Flt3- CD115lo cells =&#62; GPs, 
		Flt3- CD115hi cells =&#62; MPs+cMoPs.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Akashi, K., Traver, D., Miyamoto, T., Weissman, I. L. <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+common+myeloid+progenitor+that+gives+rise+to+all+myeloid+lineages.">A clonogenic common myeloid progenitor that gives rise to all myeloid lineages.</a> Nature. 404 (6774), 193-197 (2000).</li><li>Y&#225;&#241;ez, A., Ng, M. Y., Hassanzadeh-Kiabi, N., Goodridge, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IRF8+acts+in+lineage-committed+rather+than+oligopotent+progenitors+to+control+neutrophil+vs+monocyte+production.">IRF8 acts in lineage-committed rather than oligopotent progenitors to control neutrophil vs monocyte production.</a> Blood. 125 (9), 1452-1459 (2015).</li><li>Auffray, C., 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=CX3CR1++CD115++CD135++common+macrophage/DC+precursors+and+the+role+of+CX3CR1+in+their+response+to+inflammation.">CX3CR1+ CD115+ CD135+ common macrophage/DC precursors and the role of CX3CR1 in their response to inflammation.</a> Journal of Experimental Medicine. 206 (3), 595-606 (2009).</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 (5757), 83-87 (2006).</li><li>Onai, N., 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=Identification+of+clonogenic+common+Flt3+M-CSFR++plasmacytoid+and+conventional+dendritic+cell+progenitors+in+mouse+bone+marrow.">Identification of clonogenic common Flt3+M-CSFR+ plasmacytoid and conventional dendritic cell progenitors in mouse bone marrow.</a> Nature Immunology. 8 (11), 1207-1216 (2007).</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> Nature Immunology. 14 (8), 821-830 (2013).</li><li>Y&#225;&#241;ez, A., 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=Granulocyte-monocyte+progenitors+and+monocyte-dendritic+cell+progenitors+independently+produce+functionally+distinct+monocytes.">Granulocyte-monocyte progenitors and monocyte-dendritic cell progenitors independently produce functionally distinct monocytes.</a> Immunity. 47 (5), 890-902 (2017).</li><li>Olsson, A., 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=Single-cell+analysis+of+mixed-lineage+states+leading+to+a+binary+cell+fate+choice.">Single-cell analysis of mixed-lineage states leading to a binary cell fate choice.</a> Nature. 537 (7622), 698-702 (2016).</li><li>Amend, S. R., Valkenburg, K. C., Pienta, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+hind+limb+long+bone+dissection+and+bone+marrow+isolation.">Murine hind limb long bone dissection and bone marrow isolation.</a> Journal of Visualized Experiments. (110), (2016).</li><li>Y&#225;&#241;ez, A., Goodridge, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon+regulatory+factor+8+and+the+regulation+of+neutrophil,+monocyte,+and+dendritic+cell+production.">Interferon regulatory factor 8 and the regulation of neutrophil, monocyte, and dendritic cell production.</a> Current Opinion in Hematology. 23 (1), 11-17 (2016).</li><li>Manz, M. G., Miyamoto, T., Akashi, K., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+isolation+of+human+clonogenic+common+myeloid+progenitors.">Prospective isolation of human clonogenic common myeloid progenitors.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (18), 11872-11877 (2002).</li></ol>

The authors have nothing to disclose.

The Weissman gating strategy for mouse myeloid progenitor identification1 has been the gold standard for immunologists and hematologists for nearly 20 years, but it is now apparent that the &#34;CMP&#34; and &#34;GMP&#34; gates are very heterogeneous and more precise gating strategies are needed. The protocol that we have described here permits the identification of oligopotent and lineage-committed subsets in C57BL/6J mice for more precise quantification of specific myeloid progenitors and mappin...

Monocytes, neutrophils, and dendritic cells (DCs) are myeloid cells that arise from hematopoietic progenitors, primarily in the bone marrow, by a process called myelopoiesis. Common myeloid progenitors (CMPs) have the potential to produce myeloid cells, as well as megakaryocytes and erythrocytes, but not lymphoid cells. Granulocyte-monocyte progenitors (GMPs), which are derived from CMPs, produce granulocytes and monocytes, but have lost megakaryocyte and erythrocyte potential. Monocytes and classical and plasmacytoid DCs (cDCs/pDCs) are also thought to arise from common progenitors known as monocyte-DC progenitors (MDPs), which are produced by CMPs. Gradual restriction of lineage potential ultimately results in lineage-committed progenitors: granulocyte progenitors, monocyte progenitors, and dendritic cell progenitors (Figure 1).
Weissman and colleagues reported that CMPs are found in the Lin- c-Kit+ Sca-1- (LKS-) CD34+ Fc&#947;Rlo fraction of mouse bone marrow, while GMPs are contained in the LKS- CD34+ Fc&#947;Rhi fraction1. However, these &#34;CMP&#34; and &#34;GMP&#34; fractions are very heterogeneous. For instance, the &#34;GMP&#34; fraction also contains lineage-committed granulocyte progenitors and monocyte progenitors1,2. MDPs were separately reported to be CX3CR1+ Flt3+ CD115+ progenitors that also express CD34 and Fc&#947;R3,4. MDPs give rise to cDC/pDC-producing common DC progenitors (CDPs), which have been reported to express lower levels of c-Kit (CD117) and are not included in the LKS- fraction5.
It was previously assumed that monocytes arise via a single pathway (CMP-GMP-MDP-monocyte). Consistent with this model, monocyte-committed progenitors produced by GMPs (named monocyte progenitors, MPs)2 and MDPs (named common monocyte progenitors, cMoPs)6 appear to be the same cells on the basis of shared surface marker expression. However, we recently demonstrated that monocytes are produced independently by GMPs and MDPs, and were able to distinguish between MPs and cMoPs by single-cell RNA sequencing7.
We recently modified the Weissman &#34;CMP&#34; and &#34;GMP&#34; gating strategy to identify 6 subfractions of C57BL/6J mouse bone marrow containing different oligopotent and lineage-committed myeloid progenitor subsets. We first reported that staining for Ly6C and CD115 permits the isolation of oligopotent GMPs, as well as granulocyte progenitors (GPs) and monocyte progenitors (both MPs and cMoPs, which we are currently unable to separate) from the &#34;GMP&#34; fraction2 (LKS- CD34+ Fc&#947;Rhi gate; Figure 1). We subsequently demonstrated that MDPs are predominantly found in the &#34;CMP&#34; fraction (LKS- CD34+ Fc&#947;Rlo gate), which also contains Flt3+ CD115lo and Flt3- subsets7 (Figure 1). The CMP-Flt3+ CD115lo fraction yields both GMPs and MDPs upon adoptive transfer. The CMP-Flt3- subset contains progenitors that appear to be intermediates between CMP-Flt3+ CD115lo cells and GMPs. Unlike MDPs, both the CMP-Flt3+ CD115lo and CMP-Flt3- fractions also possess megakaryocyte and erythrocyte potential.
It is important to note, however, that it is currently unclear whether the &#34;CMP&#34; fractions contain progenitors that are truly oligopotent (e.g., individual cells within the CMP-Flt3+ CD115lo fraction that possess neutrophil, monocyte, DC, megakaryocyte, and erythrocyte potential), or alternatively, comprise a mixture of progenitors with more restricted lineage potential. Colony forming assays (methylcellulose cultures) revealed cells with granulocyte (neutrophil), erythrocyte, monocyte and megakaryocyte potential (GEMM cells) in the &#34;CMP&#34;, CMP-Flt3+ CD115lo and CMP-Flt3- fractions1,7, but do not permit the assessment of DC potential. In contrast, colony forming assays demonstrated the existence of oligopotent GMPs (progenitors with both neutrophil and monocyte potential) in the &#34;GMP&#34; fraction1,2, and this is supported by recent single-cell transcriptomic analysis8. It is not currently known, however, whether these oligopotent GMPs also produce other granulocytes (eosinophils, basophils, and mast cells).
Based on these studies, we now demonstrate how 7 surface markers (c-Kit, Sca-1, CD34, Fc&#947;R, Flt3, Ly6C and CD115) can be used to identify and isolate these 6 subsets of oligopotent and lineage-committed myeloid progenitors. The protocol described here can be applied for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments in mice, and molecular analysis (bulk and single-cell RNA sequencing, Western blotting, etc.).
The protocol consists of 3 stages: 1) preparation of a single cell suspension of bone marrow cells, 2) enrichment for hematopoietic progenitors (magnetic-activated cell sorting), and 3) identification, and isolation if desired, of progenitor subsets by flow cytometry (using an analyzer or a sorter, as appropriate). The first step is the isolation of bone marrow cells from the femurs and tibias of euthanized mice and is similar to other previously described protocols9. Next, the sample is enriched for stem and progenitor cells using a cocktail of antibodies against cell surface markers of erythrocytes, neutrophils, monocytes, lymphocytes, etc., to deplete the differentiated cells. This is not mandatory, but strongly recommended to optimize detection of the progenitor subsets, and to reduce the quantity of antibodies needed for progenitor identification and the time required for flow cytometry. The lineage depletion protocol below describes Magnetic-Activated Cell Sorting (MACS) using a Mouse Lineage Cell Depletion Kit (which contains biotinylated antibodies against CD5, CD45R (B220), CD11b, Gr-1 (Ly6G/C), 7-4, and Ter-119, plus anti-biotin microbeads) and an automated magnetic separator. The final step is the identification (and sorting, if desired) of the progenitor subsets by flow cytometry. The antibody panel described below (see also Table 1) has been designed to be used in a flow cytometer (analyzer or sorter) with 4 lasers (405 nm, 488 nm, 561 nm, 640 nm).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58061/58061fig1.jpg" /> 
Figure 1: Neutrophil, monocyte and DC progenitors and differentiation pathways. The recently revised model of myelopoiesis7 is illustrated, with the Weissman gates for &#34;CMPs&#34; (blue) and &#34;GMPs&#34; (green)1 overlaid. This figure has been modified from Y&#225;&#241;ez et al. 20177. <a href="https://www.jove.com/files/ftp_upload/58061/58061fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="945">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Mouse: Wild-type C57BL/6J (CD45.2)</td>
			<td style="width:177px;">The Jackson Laboratories</td>
			<td style="width:172px;">Cat#JAX:000664</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Lineage Cell Depletion Kit, mouse</td>
			<td style="width:177px;">Miltenyi Biotec</td>
			<td style="width:172px;">Cat#130-090-858</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Rat anti-mouse CD34 (clone RAM34) FITC</td>
			<td style="width:177px;">BD Biosciences</td>
			<td style="width:172px;">Cat#553733</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:357px;">Rat anti-mouse CD16/CD32 (Fc&#947;R; clone 93) APC-Cy7</td>
			<td style="width:177px;">BioLegend</td>
			<td style="width:172px;">Cat#101327</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:357px;">Rat anti-mouse Ly6A/E (Sca-1; clone 108113) PE-Cy7</td>
			<td style="width:177px;">BioLegend</td>
			<td style="width:172px;">Cat#108114</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:357px;">Rat anti-mouse CD117 (c-Kit; clone 2B8) Pacific Blue</td>
			<td style="width:177px;">BioLegend</td>
			<td style="width:172px;">Cat#105820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Rat anti-mouse Ly6C (clone HK1.4) PerCP-Cy5.5</td>
			<td style="width:177px;">BioLegend</td>
			<td style="width:172px;">Cat#128012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Rat anti-mouse CD115 (clone AFS98) PE</td>
			<td style="width:177px;">BioLegend</td>
			<td style="width:172px;">Cat#135506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Rat anti-mouse CD135 (Flt3; clone A2F10.1) APC</td>
			<td style="width:177px;">BD Biosciences</td>
			<td style="width:172px;">Cat#560718</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">CountBright Absolute Counting Beads</td>
			<td style="width:177px;">Thermo Fisher Scientific</td>
			<td style="width:172px;">Cat#C36950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">AutoMACS Separator</td>
			<td style="width:177px;">Miltenyi Biotec</td>
			<td style="width:172px;">N/A</td>
			<td>Use the &#34;deplete&#34; program</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">BD LSRFortessa</td>
			<td style="width:177px;">BD Biosciences</td>
			<td style="width:172px;">N/A</td>
			<td>5 lasers, 15 colors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">BD FACS Aria III cell sorter</td>
			<td style="width:177px;">BD Biosciences</td>
			<td style="width:172px;">N/A</td>
			<td>5 lasers, 13 colors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">FlowJo</td>
			<td style="width:177px;">FlowJo, LLC</td>
			<td style="width:172px;">https://www.flowjo.com</td>
			<td>For further analysis of the .fcs files</td>
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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.

Using the protocol described above, it is possible to obtain ~100 million cells (including red blood cells, or ~50 million nucleated cells) from both femurs and tibias (2 legs) of one C57BL/6J mouse (6 - 8 weeks old, male or female). 1 - 2 million Lin- cells can be isolated per mouse by MACS depletion of Lin+ cells.
Each of the 6 myeloid progenitor subsets constitutes ~1 - 4% of the Lin- cells. L...

Watch this Scientific Journal Video about Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow at JoVE.com

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

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 ...

identification-isolation-oligopotent-lineage-committed-myeloid

Research

JoVE Journal

Immunology and Infection

8.4K Views.  Cedars-Sinai Medical Center. 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.

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

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Bone Marrow-derived Macrophage Production

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

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

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

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

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

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

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

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

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

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

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

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

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

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

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

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 ...