Name: identification and isolation of oligopotent and lineage-committed myeloid progenitors from mouse bone marrow
Uploaded: 2018-07-29T15:00:00
Description: Watch this Scientific Journal Video about Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow at JoVE.com

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 to...

<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 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>
		</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. 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 ...

This method could be helpful for hematologists and immunologists studying the production and function of myeloid cells, including neutrophils, monocytes, and dendritic cells. The main advantage of this technique is that it enables more precise identification of myeloid progenitor subsets than previous strategies. To enrich bone marrow cells for progenitors by magnetic-activated cell sorting, or MACS, pellet the cells by centrifugation and resuspend the pellet in 40 microliters of MACS staining buffer per one times 10 to the seven bone marrow cells.To deplete differentiated lineage positive cells, add 10 microliters of biotin antibody cocktail per one times 10 to the seven cells and to mix by pipetting for a 10 minute incubation at four degrees Celsius. At the end of the incubation, add 30 microliters of staining buffer per one times 10 to the seven cells with mixing, followed by 20 microliters of anti-biotin microbeads per one times 10 to the seven cells. After 15 minutes at four degrees Celsius, wash the cells with one milliliter of staining buffer per one times 10 to the seven cells and resuspend the pellet in 500 microliters of fresh staining buffer for up to one times 10 to the eight cells.Next, set up the automated magnetic separator according to the manufacturer's instructions and place the tube of labeled cells into the separator. Begin the negative selection program to collect the negative fraction which contains the progenitor enriched lineage negative cells. Pellet the lineage negative cells by centrifugation and resuspend the pellet, which is now white, in two milliliters of staining buffer per mouse for counting.To isolate the myeloid progenitor cells by FACS, add one times 10 to the fifth lineage negative cells to each of eight control microcentrifuge tubes for voltage selection and color compensation and add the rest of the cell sample to a ninth microcentrifuge tube for staining with all seven of the surface marker antibodies of interest for progenitor identification and sorting. Pellet the cells by centrifugation and resuspend the control pellets in 100 microliters of FACS staining buffer and the experimental sample pellet in 100 microliters of staining buffer per five times 10 to the six cells. Next, add anti-CD16/CD32 antibody to the Fc gamma receptor single stain tube and the sample tube.Vortex gently and incubate the samples for 10 minutes at four degrees Celsius. At the end of the incubation, add the other antibodies to the appropriate corresponding single stain tubes and to the sample tube and after gentle vortexing, incubate the samples for 15 minutes at four degrees Celsius. At the end of the incubation, add 900 microliters of staining buffer to the control tubes and one milliliter of staining buffer per one times 10 to the seven cells to the sample tube for centrifugation.Resuspend the control pellets in 200 microliters of fresh staining buffer per tube and the experimental pellet in 500 microliters per 2.5 times 10 to the seven cells and transfer the labeled cells to individual corresponding five milliliter FACS tubes. Then set up the flow cytometer according to standard protocols and use the unstained and single stained controls to set the voltages and the color compensations. To identify and isolate the myeloid progenitor cells, load the experimental cell sample onto the cytometer and create a forward scatter area versus side scatter area plot, gating to exclude the debris and dead cells.Create a forward scatter height versus forward scatter width plot of the live cells and gate to exclude the doublets. Repeat this process with a side scatter height versus side scatter width plot of the forward scatter singlets and gate to exclude the doublets. Create a Sca-1 versus c-Kit plot and gate to select the c-Kit positive Sca-1 negative progenitors.Then create a CD-34 versus FC gamma receptor plot and gate to select the mixed common myeloid progenitor and mixed granulocyte monocyte progenitor populations. It's important to gate the mixed common myeloid progenitor and mixed granulocyte monocyte progenitor populations as precisely as possible. It can be helpful to use a pseudo-color density plot or a contour plot for more accurate gating.Use the cells in the mixed common myeloid progenitor gate to create a CD115 versus Flt3 plot and gate to select the common myeloid progenitor Flt3 positive CD115 low cells, the common myeloid progenitor Flt3 negative CD115 low cells, and the Flt3 positive CD115 high monocyte dendritic cell progenitors. Use the cells in the mixed granulocyte monocyte progenitor gate to create a Ly6C versus FC Gamma receptor plot and gate to select the Ly6C negative cells and the Ly6C positive cells. Then create a CD115 versus Flt3 plot for each gated mixed granulocyte monocyte progenitor cell subpopulation and gate the Ly6C negative Flt3 negative CD115 low granulocyte monocyte progenitors, the Ly6C positive Flt3 negative CD115 low granulocyte progenitors, and the Ly6C positive Flt3 negative CD115 high monocyte progenitors.Max lineage depletion is efficient at depleting differentiated cells and for enriching for the c-Kit positive progenitor cells. The lineage negative fraction does still contain some c-Kit negative cells, but these cells will be eliminated in subsequent flow cytometry gating steps. Progenitor yields after FACS sorting can vary depending on the sorter settings used, but it should be possible to obtain between one to four times 10 to the fourth cells per fraction from each mouse with a post-sort purity of greater than 95%for each fraction.Good staining is critical for clean separation of the populations. You may need to titrate the antibodies or choose different fluorophores to ensure optimal separation. This protocol can be used to identify progenitor cells to assess differences in bone marrow composition between samples, to isolate progenitors for molecular analysis or to assess their ability to produce myeloid cells.

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