This work is supported by NIH grants R01DK130498, R01DK120639, and R01HL141402

All experiments were conducted in accordance with animal protocols A-1586 and 202200017 approved by the University of Massachusetts Chan Medical School Institutional Animal Care and Use Committee.
NOTE: Two protocols are detailed here: first, flow cytometric analysis (section 1), followed by protocol adjustments for flow cytometric sorting (section 2). The protocol below uses a flow cytometer/sorter with 10 channels. An example setup is provided in Table 1, referred to in step...

<ol>
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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=Population+snapshots+predict+early+haematopoietic+and+erythroid+hierarchies.">Population snapshots predict early haematopoietic and erythroid hierarchies.</a> Nature. 555 (7694), 54-60 (2018).</li><li>Socolovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+specialized+cell+cycles+during+erythroid+lineage+development:+Insights+from+single-cell+RNA+sequencing.">The role of specialized cell cycles during erythroid lineage development: Insights from single-cell RNA sequencing.</a> International Journal of Hematology. 116 (2), 163-173 (2022).</li><li>Hwang, Y., Hidalgo, D., Socolovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+shifting+shape+and+functional+specializations+of+the+cell+cycle+during+lineage+development.">The shifting shape and functional specializations of the cell cycle during lineage development.</a> WIREs Mechanisms of Disease. 13 (2), 1504 (2021).</li><li>Axelrad, A. A., McLeod, D. L., Shreeve, M. M., Heath, D. S., Robinson, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+Cells+that+Produce+Erythrocytic+Colonies+In+Vitro.">Properties of Cells that Produce Erythrocytic Colonies In Vitro.</a> Hemopoiesis in Culture. , (1974).</li><li>Heath, D. S., Axelrad, A. A., McLeod, D. L., Shreeve, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+the+erythropoietin-responsive+progenitors+BFU-E+and+CFU-E+in+mouse+bone+marrow+by+unit+gravity+sedimentation.">Separation of the erythropoietin-responsive progenitors BFU-E and CFU-E in mouse bone marrow by unit gravity sedimentation.</a> Blood. 47 (5), 777-792 (1976).</li><li>Iscove, N. N., Sieber, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythroid+progenitors+in+mouse+bone+marrow+detected+by+macroscopic+colony+formation+in+culture.">Erythroid progenitors in mouse bone marrow detected by macroscopic colony formation in culture.</a> Experimental Hematology. 3 (1), 32-43 (1975).</li><li>Gregory, C. J., McCulloch, E. A., Till, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythropoietic+progenitors+capable+of+colony+formation+in+culture:+State+of+differentiation.">Erythropoietic progenitors capable of colony formation in culture: State of differentiation.</a> Journal of Cell Physiology. 81 (3), 411-420 (1973).</li><li>Gregory, C. J., Tepperman, A. D., McCulloch, E. A., Till, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythropoietic+progenitors+capable+of+colony+formation+in+culture:+Response+of+normal+and+genetically+anemic+W-W-V+mice+to+manipulations+of+the+erythron.">Erythropoietic progenitors capable of colony formation in culture: Response of normal and genetically anemic W-W-V mice to manipulations of the erythron.</a> Journal of Cell Physiology. 84 (1), 1-12 (1974).</li><li>Gregory, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythropoietin+sensitivity+as+a+differentiation+marker+in+the+hemopoietic+system:+studies+of+three+erythropoietic+colony+responses+in+culture.">Erythropoietin sensitivity as a differentiation marker in the hemopoietic system: studies of three erythropoietic colony responses in culture.</a> Journal of Cell Physiology. 89 (2), 289-301 (1976).</li><li>Pop, R., 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+key+commitment+step+in+erythropoiesis+is+synchronized+with+the+cell+cycle+clock+through+mutual+inhibition+between+PU.1+and+S-phase+progression.">A key commitment step in erythropoiesis is synchronized with the cell cycle clock through mutual inhibition between PU.1 and S-phase progression.</a> PLoS Biology. 8 (9), 1000484 (2010).</li><li>Koulnis, M., 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+and+analysis+of+mouse+erythroid+progenitors+using+the+CD71/TER119+flow-cytometric+assay.">Identification and analysis of mouse erythroid progenitors using the CD71/TER119 flow-cytometric assay.</a> Journal of Visualized Experiments. (54), e2809 (2011).</li><li>Liu, Y., 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=Suppression+of+Fas-FasL+coexpression+by+erythropoietin+mediates+erythroblast+expansion+during+the+erythropoietic+stress+response+in+vivo.">Suppression of Fas-FasL coexpression by erythropoietin mediates erythroblast expansion during the erythropoietic stress response in vivo.</a> Blood. 108 (1), 123-133 (2006).</li><li>Socolovsky, M., 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=Ineffective+erythropoiesis+in+Stat5a(-/-)5b(-/-)+mice+due+to+decreased+survival+of+early+erythroblasts.">Ineffective erythropoiesis in Stat5a(-/-)5b(-/-) mice due to decreased survival of early erythroblasts.</a> Blood. 98 (12), 3261-3273 (2001).</li><li>Chen, 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=Resolving+the+distinct+stages+in+erythroid+differentiation+based+on+dynamic+changes+in+membrane+protein+expression+during+erythropoiesis.">Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression during erythropoiesis.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (41), 17413-17418 (2009).</li><li>Hwang, Y., Hidalgo, D., Socolovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+shifting+shape+and+functional+specializations+of+the+cell+cycle+during+lineage+development.">The shifting shape and functional specializations of the cell cycle during lineage development.</a> WIREs Mechanisms of Disease. 13 (2), 1504 (2020).</li><li>Hidalgo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EpoR+stimulates+rapid+cycling+and+larger+red+cells+during+mouse+and+human+erythropoiesis.">EpoR stimulates rapid cycling and larger red cells during mouse and human erythropoiesis.</a> Nature Communications. 12 (1), 7334 (2021).</li><li>Porpiglia, E., Hidalgo, D., Koulnis, M., Tzafriri, A. R., Socolovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stat5+signaling+specifies+basal+versus+stress+erythropoietic+responses+through+distinct+binary+and+graded+dynamic+modalities.">Stat5 signaling specifies basal versus stress erythropoietic responses through distinct binary and graded dynamic modalities.</a> PLoS Biology. 10 (8), 1001383 (2012).</li><li>Koulnis, M., 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=Contrasting+dynamic+responses+in+vivo+of+the+Bcl-xL+and+Bim+erythropoietic+survival+pathways.">Contrasting dynamic responses in vivo of the Bcl-xL and Bim erythropoietic survival pathways.</a> Blood. 119 (5), 1228-1239 (2012).</li><li>Shearstone, J. R., 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=Global+DNA+demethylation+during+mouse+erythropoiesis+in+vivo.">Global DNA demethylation during mouse erythropoiesis in vivo.</a> Science. 334 (6057), 799-802 (2011).</li><li>Koulnis, M., Liu, Y., Hallstrom, K., Socolovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+autoregulation+by+Fas+stabilizes+adult+erythropoiesis+and+accelerates+its+stress+response.">Negative autoregulation by Fas stabilizes adult erythropoiesis and accelerates its stress response.</a> PLoS One. 6 (7), 21192 (2011).</li><li>Pronk, C. 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=Elucidation+of+the+phenotypic,+functional,+and+molecular+topography+of+a+myeloerythroid+progenitor+cell+hierarchy.">Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy.</a> Cell Stem Cell. 1 (4), 428-442 (2007).</li><li>Dolznig, H., 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=Expansion+and+differentiation+of+immature+mouse+and+human+hematopoietic+progenitors.">Expansion and differentiation of immature mouse and human hematopoietic progenitors.</a> Methods in Molecular Medicine. 105, 323-344 (2005).</li><li>Li, 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=Isolation+and+transcriptome+analyses+of+human+erythroid+progenitors:+BFU-E+and+CFU-E.">Isolation and transcriptome analyses of human erythroid progenitors: BFU-E and CFU-E.</a> Blood. 124 (24), 3636-3645 (2014).</li><li>Zhang, J., Socolovsky, M., Gross, A. W., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Ras+signaling+in+erythroid+differentiation+of+mouse+fetal+liver+cells:+Functional+analysis+by+a+flow+cytometry-based+novel+culture+system.">Role of Ras signaling in erythroid differentiation of mouse fetal liver cells: Functional analysis by a flow cytometry-based novel culture system.</a> Blood. 102 (12), 3938-3946 (2003).</li><li>Flygare, J., Rayon Estrada, V., Shin, C., Gupta, S., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF1alpha+synergizes+with+glucocorticoids+to+promote+BFU-E+progenitor+self-renewal.">HIF1alpha synergizes with glucocorticoids to promote BFU-E progenitor self-renewal.</a> Blood. 117 (12), 3435-3444 (2011).</li><li>Pronk, C. J., Attema, J., Rossi, D. 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The ability to prospectively isolate BFU-e and CFU-e progenitors directly from fresh tissue with high purity had previously eluded investigators. Our novel approach, validated using scRNAseq and cell fate assays1,27, now offers the tools to do this.
There are a number of key points for successfully executing both the sorting and the analytical protocols. First, the cells need to be spun at 900 x g to prevent the loss of low-de...

Erythropoiesis may be divided into two principal phases: early erythropoiesis and erythroid terminal differentiation (Figure 1)1,2,3. In early erythropoiesis, hematopoietic stem cells commit to the erythroid lineage and give rise to early erythroid progenitors, which were first identified in the 1970s based on their colony-forming potential in semi-solid medium4,5,6,7,8,9. Broadly, erythroid progenitors are divided into two categories: earlier progenitors that each give rise to a &#34;burst&#34; (a large aggregate of smaller erythroid cell clusters), named &#34;burst-forming unit erythroid&#34; or BFU-e4,5,6; and their progeny, which each form a single, small erythroid cell cluster or colony, named &#34;colony-forming unit erythroid&#34; or CFU-e7,8,9. BFU-e and CFU-e do not yet express terminal erythroid genes and are not morphologically recognizable. After a number of self-renewal or expansion cell divisions, the CFU-e undergoes a transcriptional switch in which erythroid genes such as globins are induced, thereby transitioning into erythroid terminal differentiation (ETD)1,10. During ETD, erythroblasts undergo three to five maturational cell divisions before enucleating to form reticulocytes, which mature into red cells.
Erythroblasts during terminal differentiation were originally classified based on their morphology into proerythroblasts, basophilic, polychromatic, and orthochromatic. The advent of flow cytometry allowed their prospective sorting and isolation based on cell size (measured by forward scatter, FSC) and two cell surface markers, CD71 and Ter11911,12,13 (Figure 1). This and similar flow cytometric approaches14 have revolutionized the investigation of the molecular and cellular aspects of ETD, allowing developmental stage-specific analysis of erythroblasts in vivo and in vitro10,15,16,17,18,19,20. The CD71/Ter119 approach is now used routinely in the analysis of erythroid precursors.
Until recently, a similar, accessible flow cytometric approach for direct, high-purity prospective isolation of CFU-e and BFU-e from mouse tissue has eluded investigators. Instead, investigators have used flow cytometric strategies that isolate only a fraction of these progenitors, often in the presence of non-erythroid cells that co-purify within the same flow cytometric subsets21. Consequently, the investigation of BFU-e and CFU-e was limited to in vitro differentiation systems that derive and amplify BFU-e and CFU-e from earlier bone marrow progenitors. It is then possible to apply flow cytometric strategies that distinguish CFU-e from BFU-e in these erythroid progenitor-enriched cultures22,23. An alternative approach makes use of fetal CFU-e and BFU-e, which are highly enriched in the Ter119-negative fraction of the mouse fetal liver at mid gestation10,24,25. Neither of these approaches, however, allow the investigation of adult BFU-e and CFU-e in their physiological state in vivo. The magnitude of the challenge may be appreciated when recalling that, based on colony formation assays, these cells are present in the adult bone marrow at a frequency of only 0.025% and 0.3%, respectively6.
The protocol described here is a novel flow cytometric approach based on single-cell transcriptomic analysis of freshly harvested Kit+ mouse bone marrow cells (Kit is expressed by all of the early progenitor populations of the bone marrow)1. Our approach contains some cell surface markers that were already in use by Pronk et al.21,26. Single-cell transcriptomes were used to determine combinations of cell surface markers that identify erythroid and other early hematopoietic progenitors (Figure 2). Specifically, the CD55+ fraction of lineage-negative (Lin-) Kit+ cells may be subdivided into five populations, three of which yield contiguous segments of the erythroid trajectory (Figure 2). The transcriptomic identities of each of these populations were confirmed by sorting, followed by scRNAseq and projection of the sorted single-cell transcriptomes back onto the original transcriptomic map (the gene expression in each of the five populations and the entire bone-marrow dataset can be explored in https://kleintools.hms.harvard.edu/paper_websites/tusi_et_al/index.html)1.&#160;The cell fate potential of each of the populations was confirmed using traditional colony formation assays (Figure 2), as well as a novel high-throughput single-cell fate assay1,27. These analyses show that the novel flow cytometric approach results in high-purity isolation of all the BFU-e and CFU-e progenitors of fresh adult bone marrow and spleen. Specifically, population 1 (P1) contains only CFU-e and no other hematopoietic progenitors, and population 2 (P2) contains all of the bone marrow&#39;s BFU-e progenitors and a small number of CFU-e but no other progenitors1. The detailed protocol below is further illustrated with an example experiment in mice that were injected with either saline or with the erythropoiesis-stimulating hormone erythropoietin (Epo).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 M EDTA, pH 8.0</td>
			<td>Life Technologies</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;L large orifice tips</td>
			<td>USA sceintific</td>
			<td>1011-9000</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 anti-mouse CD55 (DAF) Antibody</td>
			<td>BioLegend</td>
			<td>131806</td>
			<td></td>
		</tr>
		<tr>
			<td>APC/Cyanine7 anti-mouse CD117 (c-kit) Antibody</td>
			<td>BioLegend</td>
			<td>105826</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-CD11b</td>
			<td>BD Biosciences</td>
			<td>557395</td>
			<td>M1/70 (clone)</td>
		</tr>
		<tr>
			<td>Biotin-CD19</td>
			<td>BD Biosciences</td>
			<td>553784</td>
			<td>1D3 (clone)</td>
		</tr>
		<tr>
			<td>Biotin-CD4</td>
			<td>BD Biosciences</td>
			<td>BDB553045</td>
			<td>RM4-5 (clone)</td>
		</tr>
		<tr>
			<td>Biotin-CD8a</td>
			<td>BD Biosciences</td>
			<td>BDB553029</td>
			<td>53-6.7 (clone)</td>
		</tr>
		<tr>
			<td>Biotin-F4/80</td>
			<td>Biolegend</td>
			<td>123106</td>
			<td>BM8 (clone)</td>
		</tr>
		<tr>
			<td>Biotin-Ly-6G and Ly-6C</td>
			<td>BD Biosciences</td>
			<td>553125</td>
			<td>RB6-8C5 (clone)</td>
		</tr>
		<tr>
			<td>Biotin-TER-119</td>
			<td>BD Biosciences</td>
			<td>553672</td>
			<td>TER-119 (clone)</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma aldritch</td>
			<td>A1470</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 421 anti-human/mouse CD49f Antibody</td>
			<td>BioLegend</td>
			<td>313624</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 605 anti-mouse CD41 Antibody</td>
			<td>BioLegend</td>
			<td>133921</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 650 anti-mouse CD150 (SLAM) Antibody</td>
			<td>BioLegend</td>
			<td>115931</td>
			<td></td>
		</tr>
		<tr>
			<td>BUV395 Rat Anti-Mouse TER-119/Erythroid Cells</td>
			<td>BD Biosciences</td>
			<td>563827</td>
			<td></td>
		</tr>
		<tr>
			<td>ChromPure Rabbit IgG, whole molecule</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>011-000-003</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Life Technologies</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital DIVA hardware and software for LSR II</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse F4/80 Antibody</td>
			<td>BioLegend</td>
			<td>123108</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Rat Anti-CD11b</td>
			<td>BD Biosciences</td>
			<td>557396</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Rat Anti-Mouse CD19</td>
			<td>BD Biosciences</td>
			<td>553785</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Rat Anti-Mouse CD4</td>
			<td>BD Biosciences</td>
			<td>553047</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Rat Anti-Mouse CD8a</td>
			<td>BD Biosciences</td>
			<td>553031</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Rat Anti-Mouse Ly-6G and LY-6C</td>
			<td>BD Biosciences</td>
			<td>553127</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo software&#160;</td>
			<td>FlowJo</td>
			<td>version 10</td>
			<td>Flow cytometer analysis software</td>
		</tr>
		<tr>
			<td>LSR II digital multiparameter flow cytometer analyzer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometer&#160;</td>
		</tr>
		<tr>
			<td>NewlineNY Stainless Steel Hand Masher &#38; Bowl, Mortar and Pestle Set</td>
			<td>Amazon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal rat serum</td>
			<td>Stem Cell Technologies</td>
			<td>13551</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse CD105 Antibody</td>
			<td>BioLegend</td>
			<td>120408</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cyanine7 anti-mouse CD71 Antibody</td>
			<td>BioLegend</td>
			<td>113812</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline, 10x Solution</td>
			<td>Fisher scientific</td>
			<td>BP3994</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin Nanobeads</td>
			<td>BioLegend</td>
			<td>480016</td>
			<td>Magnetic beads</td>
		</tr>
	</tbody>
</table>

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.

The protocol describes a flow cytometric approach to identify BFU-es and CFU-es in freshly harvested bone marrow and spleen cells. It starts with harvesting fresh BM and spleen from mice and immediately placing the tissue on ice. All procedures are conducted in the cold to preserve cell viability. Cells are labeled with a &#34;lineage&#34; antibody cocktail that allows the exclusion of all cells expressing markers of differentiated blood lineages (the FITC- Lin cocktail, Table 3, in the case of flow cyto...

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

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 ...

This protocol allows for the identification and isolation of BFU-E and CFU-E erythroid progenitors directly from freshly-harvested mouse tissue like bone marrow and spleen. Using this technique, we can isolate pure populations of erythroid progenitors, which was not possible with the previous flow protocols. This method greatly enhances our ability to study erythroid disease mouse models by allowing progenitor isolation for many downstream experiments.To begin, place the freshly dissected femurs and tibia directly into cold staining buffer or SB-5 and keep the tubes on ice until ready to extract the bone marrow. Place a clean sterilized mortar on ice in a bucket and transfer the bones to the mortar. Using the dissection scissors, snip off approximately 1-millimeter ends of each bone.Use a 3-milliliter syringe equipped with a 26 gauge needle containing SB-5 to flush the marrow out of the bones and collect it into the mortar and repeat the process 3 to 5 times using fresh buffer each time until the bones appear colorless. Filter the flushed marrow through a 100-micrometer mesh and collect the cells in a 50-milliliter centrifuge tube placed on ice. Next, use the mortar and pestle to crush the flushed bones in 5 to 10 milliliters of SB-5.Filter the mixture of crushed bones and buffer through the 100-micrometer cell strainer to pool with the previously collected cells. Set up a 100-micrometer mesh filter on an empty 50-milliliter centrifuge tube placed on ice. Place a single spleen on the mesh filter and add 0.5 to 1 milliliter of SB-5 to the spleen to ensure it does not dry.Using the rubber side of a 3 or 5-milliliter syringe plunger, mash the spleen through the mesh. Add more SB-5, 5 milliliters at a time, and continue to mash until all the cells have been collected in the tube. Prepare a single-cell suspension by pipetting up and down the collected cells with 2 milliliters of SB-5 using a large orifice tip.Stain the fluorescence minus one or FMO controls by adding all the antibodies except the FMO's namesake antibody to the cell suspension in the FACS tube. To stain the single-color controls, add a single antibody to the cell suspension in the FACS tube. Vortex each control tube for 2 to 3 seconds at 3000 RPM and then incubate at 4 degrees Celsius rocking for 2.5 hours in the dark.After the incubation, wash the cells with SB-5 and make up the volume of the cell suspension to 4 milliliters with SB-5. Spin down the cells at 900 g for 10 minutes at 4 degrees Celsius and aspirate the supernatant. Re-suspend the unstained and all the single-color controls in 300 microliters of SB-5 and filter through a 40-micrometer filter mesh into new FACS tubes.Make a DAPI SB-5 solution by diluting the DAPI stock at a ratio of 1 to 10, 000 in SB-5. Re-suspend the FMOS in 1.8 milliliters of DAPI SB-5 and DAPI single-color control in 300 microliters of DAPI SB-5, then filter them through a 40-micrometer filter mesh into new FACS tubes. After adding the antibody master mix to each sample, vortex the tubes for 2 to 3 seconds at 3000 rpm followed by incubation at 4 degrees Celsius in the dark for 2.5 hours in the rocking condition.After washing the cells as demonstrated previously, re-suspend the samples in 1.8 milliliters of DAPI SB-5 and filter through a 40-micrometer filter mesh into a new FACS tube and analyze the samples using a cystometer. Use the forward scatter to remove the debris based on size, then use the forward scatter height versus forward scatter area histogram to exclude cell aggregates and select single cells. Repeat the exclusion with the side scatter height versus side scatter area histogram.Exclude the dead cells by gating out the positive cells for DAPI. Select the lineage negative TUR one 19 negative kit positive cells. And from these select a subpopulation that expresses CD 55.Subdivide the lineage negative TUR one 19, negative kit, positive, CD 55 positive cells into 2 principle populations, P six and P seven based on the expression of CD 49 F and CD 1 0 5. Now based on the expression of CD 150 and CD 41, subdivide P 6 further into P 3, P 4 and P 5. Similarly, based on the expression of CD 150 and CD 71, subdivide P 7 further into BFUE, early CFUE and late CFUE.In the present study, burst and colony forming units were identified from the freshly harvested bone marrow and spleen cells. After 72 hours of erythropoietin, or vehicle injection in the mice, erythropoietin stimulation showed expansion of the early and late colony forming unit populations P 1 low and P 1 high in the harvested bone marrow and spleen cells. The response of bone marrow and spleen progenitors to erythropoietin in vivo also showed a higher number of cells in the early and late colony forming unit populations P 1 low and P 1 high, compared to vehicle control.It is most important to generate a single cell suspension before labeling the cells with antibodies. This can be achieved by pipetting up and down repeatedly and by including EDTA in the buffer. The purified progenitors can be used for any downstream cellular and molecular analysis.For example, single cell transcriptomics, atesque, colony assays and biochemistry. This technique helped us test the predictions from single cell RNA-seq experiments.

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2.5K 조회수.  UMASS Chan Medical School. 이 프로토콜을 사용하면 골수 및 비장과 같이 새로 수확한 마우스 조직에서 직접 BFU-E 및 CFU-E 적혈구 전구세포를 식별하고 분리할 수 있습니다. 이 기술을 사용하여 이전 흐름 프로토콜에서는 불가능했던 적혈구 전구 세포의 순수한 개체군을 분리 할 수 있습니다. 이 방법은 많은 다운스트림 실험에서 전구 분리를 허용함으로써 적혈구 질환 마우스 모델을 연구하는 능력을 ?...