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 mapping of myelopoiesis pathways, as well as investigation of molecular mechanisms operating at specific stages of myeloid differentiation. The progenitor subsets can be sorted, if desired, for molecular analyses, in vitro and in vivo assessment of differentiation etc.
The protocol was developed using young (6 - 8 weeks old) C57BL/6J mice and is suitable for both male and female mice. We have not characterized the progenitor subsets in older mice. We don&#39;t anticipate the markers will change with age, but the relative proportions of the subsets may vary. We have not assessed the progenitor subsets in other mouse strains.
The first application of our modified gating strategy was to provide mechanistic insight into the regulation of myeloid cell fate choice by the transcription factor interferon regulatory factor 8 (IRF8)2. IRF8 was previously reported to be expressed by GMPs (the Weissman &#34;GMP&#34; fraction) and required for monocyte differentiation by promoting monocyte gene expression, suppressing neutrophil gene expression, and promoting neutrophil apoptosis10. Using our modified gating strategy, we demonstrated that IRF8 is not expressed by oligopotent GMPs, but is expressed by both GPs and MPs+cMoPs2. Moreover, we demonstrated that IRF8 regulates GP and MP+cMoP survival and differentiation, rather than production of the lineage-committed progenitors by oligopotent GMPs. Similarly, we recently used our modified gating strategy to re-define the model of myelopoiesis, and demonstrated that GMPs and MDPs produce functionally distinct inflammatory monocytes via 2 independent pathways7.
The lineage depletion step can be performed using a manual magnetic separator instead of an automated magnetic separator, and lineage depletion can also be achieved by flow cytometry using the biotinylated lineage marker antibody cocktail and a fluorophore-conjugated anti-biotin antibody, with gating to select the unstained cells. Lineage depletion excludes most differentiated cells and enriches for c-Kit+ progenitors to minimize the time and costs associated with the subsequent steps (i.e., reduces antibody volumes for flow cytometry, analyzer/sorter time, etc.). Investigators should refer to the manufacturer&#39;s datasheet to determine whether the lineage depletion step worked as efficiently as anticipated for the specific kit used. However, it doesn&#39;t matter if lineage depletion is not 100% efficient because differentiated (Lin+) cells don&#39;t express c-Kit, so any differentiated cells remaining after lineage depletion (as well as other c-Kit- cells) are subsequently excluded by flow cytometry gating. It is also important to note that GPs and MPs express Ly6C but, unlike the Ly6C expressed by monocytes, the progenitor Ly6C (presumably a different isoform) is not detected by the Gr-1 antibody often included in lineage depletion kits, and thus GPs and MPs remain after lineage depletion2.
Good staining is important for all the surface markers, but particularly critical for Fc&#947;R to permit accurate gating of the Fc&#947;Rlo &#34;CMPs&#34; and the Fc&#947;Rhi &#34;GMPs&#34;. Optimal fluorophore choice and antibody dilution are essential for good separation, and an Fc&#947;R blocking reagent (commonly used prior to staining for flow cytometry) must not be used. It is also important to note that CD115 is downregulated when the cells are maintained at 4&#176;C or on ice for extended periods, so it is important to work quickly to obtain good staining. We recommend processing the cells within 1 - 2 hours to preserve their integrity. If live cells are not required, stained cells can be fixed and acquired within 48 hours.
If the isolated fractions contain significant numbers of dead cells, it is possible to include a viability dye (e.g., propidium iodide) to more accurately gate live cells, although this would probably necessitate a change in the combination of fluorochrome-conjugated antibodies. If desired, cell counts can be calculated by adding counting beads at a known concentration to the sample for flow cytometry. Counting beads are fluorescent latex beads that can be distinguished from the cells by FSC-A and SSC-A or by their high fluorescence intensity. Cell numbers can be calculated using the following formula:
(number of cell events / number of bead events) x (number of beads added to sample / volume of sample in &#956;L) = concentration of sample as cells/&#956;L
One limitation of the current protocol is that it does not permit separate identification of MPs and cMoPs, which complicates evaluation of these progenitors, investigation of their gene expression and functional properties, etc. For example, an observed increase in MP+cMoP numbers does not indicate whether enhanced monocyte production occurs via the GMP pathway, the MDP pathway, or both. In ongoing studies, we hope to identify surface markers that distinguish between MPs and cMoPs. In the meantime, evaluation of these progenitors by single-cell RNA sequencing7 could be used to profile the relative proportions of MPs and cMoPs.
Single-cell RNA sequencing studies will likely also reveal additional heterogeneity within the 6 progenitor fractions described in this protocol, some of which will reflect different maturation states e.g., GPs that have only recently lost monocyte potential versus GPs that are poised to proceed to the next stage of differentiation. The &#34;GMP&#34; fraction subsets may also contain progenitors with the potential to produce other granulocytes (eosinophils, basophils, and mast cells), but we have not investigated this. As noted in the Introduction, it is also currently unclear whether the CMP-Flt3+ CD115lo, CMP-Flt3- and MDP fractions truly contain the predicted oligopotent progenitors or alternatively comprise a mixture of progenitors with more limited lineage potential, but single-cell RNA sequencing may provide insight into this important question.
A new approach is now required for the identification of subsets of human myeloid progenitors. Weissman and colleagues previously defined myeloid subsets (including &#34;CMPs&#34; and &#34;GMPs&#34;) in human bone marrow and cord blood11, but they are similarly heterogeneous. Unfortunately, translation of mouse gating strategies to human progenitors is complicated by notable differences in surface marker expression between human and mouse progenitors; e.g., CD34 expression is limited to myeloid progenitor subsets in the mouse, but more broadly observed on human progenitors including hematopoietic stem cells. Single-cell RNA sequencing studies should, however, also permit the identification of candidate markers to separate the human progenitor subsets.

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.remotexs.ntu.edu.sg/files/ftp_upload/58061/58061fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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. Lineage depletion is efficient at depleting differentiated cells and enriching for progenitors, but the Lin- fraction contains a large proportion of c-Kit- cells in addition to c-Kit+ progenitors (Figure 3A). Progenitor yields after FACS sorting can vary depending on the sorter settings used (e.g., maximum yield versus optimal purity), but in general it is possible to obtain 10,000 - 40,000 cells per fraction (Figure 3B). Post-sort flow cytometry analysis should reveal &#62;95% purity of each fraction (Figure 3C).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58061/58061fig2.jpg" /> 
Figure 2: Gating strategy for the identification of the 6 myeloid progenitor fractions. (A) Progressive gating of the live Lin- cells -&#62; singlets (FSC) -&#62; singlets (SSC) -&#62; LKS- cells -&#62; CD34+ Fc&#947;Rlo (&#34;CMP&#34;) and CD34+ Fc&#947;Rhi (&#34;GMP&#34;) fractions -&#62; 6 myeloid progenitor subsets. (B) Summary of surface marker expression by the 6 progenitor fractions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58061/58061fig2large.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/58061/58061fig3.jpg" /> 
Figure 3: Representative progenitor yields and purity. (A) Representative flow cytometry plots showing c-Kit and CD11b expression by bone marrow cells before and after lineage depletion, demonstrating enrichment of progenitors (c-Kit+ cells). (B) Cell yields for each progenitor fraction, presented as mean + standard deviation of 30 experiments. Bone marrow cells from up to 20 mice were pooled for each experiment. (C) Representative flow cytometry plots from post-sort analysis, demonstrating the purity of FACS-sorted progenitor fractions. Panel C has been modified from Y&#225;&#241;ez et al. 20177. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58061/58061fig3large.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>Laser</td>
			<td>Channel</td>
			<td>Filter</td>
			<td>Mirror</td>
		</tr>
		<tr>
			<td>405 nm</td>
			<td>A.</td>
			<td>670/30</td>
			<td>635LP</td>
		</tr>
		<tr>
			<td></td>
			<td>B.</td>
			<td>525/50</td>
			<td>505LP</td>
		</tr>
		<tr>
			<td></td>
			<td>C. c-Kit-Pacific Blue</td>
			<td>450/50</td>
			<td></td>
		</tr>
		<tr>
			<td>488 nm</td>
			<td>A. (FSC)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>B. Ly6C-PerCP-Cy5.5</td>
			<td>710/50</td>
			<td>690LP</td>
		</tr>
		<tr>
			<td></td>
			<td>C. CD34-FITC</td>
			<td>525/50</td>
			<td>505LP</td>
		</tr>
		<tr>
			<td></td>
			<td>D. SSC</td>
			<td>488/10</td>
			<td></td>
		</tr>
		<tr>
			<td>561 nm</td>
			<td>A. Sca-1-PE-Cy7</td>
			<td>780/40</td>
			<td>750LP</td>
		</tr>
		<tr>
			<td></td>
			<td>B.</td>
			<td>710/50</td>
			<td>690LP</td>
		</tr>
		<tr>
			<td></td>
			<td>C.</td>
			<td>660/20</td>
			<td>640LP</td>
		</tr>
		<tr>
			<td></td>
			<td>D.</td>
			<td>610/20</td>
			<td>600LP</td>
		</tr>
		<tr>
			<td></td>
			<td>E. CD115-PE</td>
			<td>582/15</td>
			<td></td>
		</tr>
		<tr>
			<td>640 nm</td>
			<td>A. Fc&#978;R-APC-Cy7</td>
			<td>780/60</td>
			<td>750LP</td>
		</tr>
		<tr>
			<td></td>
			<td>B.</td>
			<td>730/45</td>
			<td>690LP</td>
		</tr>
		<tr>
			<td></td>
			<td>C. Flt3-APC</td>
			<td>670/30</td>
			<td></td>
		</tr>
	</tbody>
</table>

Table 1: Antibody panel and flow cytometer configuration.

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.5K Views.  Cedars-Sinai Medical Center. We laten zien hoe te identificeren en te isoleren 6 deelverzamelingen van myeloïde voorlopercellen uit lymfkliertest beenmerg met behulp van een combinatie van magnetische en fluorescentie sorteren (MACS en FACS). Dit protocol kan worden gebruikt voor in vitro cultuur assays (methylcellulose of vloeistof culturen), in vivo adoptief overdracht experimenten en RNA/eiwit analyses.

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

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Developmental Biology

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

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 ...

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

Isolation and Characterization of Bone Marrow Microenvironment in Myeloid Neoplasms

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

Author Spotlight: Analyzing Bone Marrow Microenvironment in Murine Hematological Malignancies 

Here a detailed protocol to isolate and characterize bone marrow microenvironmental populations from murine models of myelodysplastic syndromes and acute myeloid leukemia is presented. This technique identifies changes in the non-hematopoietic bone marrow niche, including the endothelial and mesenchymal stromal cells, with disease progression.

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and ...

Cancer Research

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 Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. 
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

Biology

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).

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 ...

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 ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

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

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