Name: identification of erythromyeloid progenitors and their progeny in the mouse embryo by flow cytometry
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Description: Watch this Scientific Journal Video about Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry at JoVE.com

The authors thank Prof Frederic Geissmann and Prof Christian Schulz for insightful discussions; Dr Hannah Garner for critical reading of the manuscript, Dr Xavier Montagutelli and Dr Jean Jaubert and the staff of the Institut Pasteur animal facility for support with mouse husbandry; and Pascal Dardenne and Vytaute Boreikaite, an Amgen Scholar, for their technical assistance. Research in the E.G.P. laboratory is funded by the Institut Pasteur, the CNRS, the Cercle FSER (FRM and a starting package from the Institut Pasteur and the REVIVE consortium. L.I. is supported by a PhD fellowship from the REVIVE consortium.

Animal procedures were performed in accordance with the approved institutional animal care and use committee of the Institut Pasteur (CETEA).
1. In Utero Pulse Labeling in Csf1r MeriCreMer Rosa LSL-YFP Embryos
<ol>
	<li>Prepare the stock solution of 4-hydroxytamoxifen (OH-TAM, 50 mg/mL).
	<ol>
		<li>Under the fumehood, open the 25 mg vial of OH-TAM and add 250 &#181;L ethanol (100%).</li>
		<li>Transfer the OH-TAM solution to a 2 mL microcentrifuge tube with a truncated tip and vortex at maximum speed for 10 min.</li>
		<li>Sonicate 30 min in a sonicator bath.</li>
		<li>Add 250 &#181;L of PEG-35 castor oil under the fumehood to obtain a 50 mg/mL stock solution. 
		NOTE: PEG-35 castor oil is a solvent with amphiphilic properties that binds hydrophobic molecules and solubilizes them in aqueous solvents.</li>
		<li>Vortex for approximately 5 min at maximum speed and sonicate 30 min in a sonicator bath.</li>
		<li>Aliquot 90 &#181;L (4.5 mg) per microcentrifuge tube (1 aliquot per injection).</li>
		<li>Store at 4 &#176;C for 1 week or at -20 &#176;C for long term as previously described 13.</li>
	</ol>
	</li>
	<li>Prepare the stock solution of Progesterone (10 mg/mL)
	<ol>
		<li>Add 250 &#181;L of 100% Ethanol to 25 mg of progesterone to prepare a 10 mg/100 &#181;L suspension under the fumehood. Vortex gently.</li>
		<li>Add 2250 &#181;L autoclaved sunflower oil to make 10 mg/ml progesterone solution under the hood.</li>
		<li>Vortex for approximately 5 min at maximum speed, aliquot and store at 4 &#176;C. Note that the solution is clear when stored.</li>
	</ol>
	</li>
	<li>Tamoxifen administration 
	NOTE: Embryonic development was estimated considering the day of vaginal plug formation as Embryonic day (E) 0.5. Recombination is induced by single injection at E8.5 into pregnant Csf1rMeriCreMerfemales 12. Supplement OH-TAM with 37.5 &#181;g per g of Progesterone to reduce abortion rates after tamoxifen administration. Inject at 1 pm (for vaginal plug observed in the morning).
	<ol>
		<li>On the morning of injection, sonicate an aliquot of the OH-TAM solution for 10 min (or until completely resuspended).</li>
		<li>Add 360 &#181;L NaCl 0.9% under the fumehood to obtain a 10 mg/mL OH-TAM solution and vortex thoroughly. Sonicate until the solution is clear and completely resuspended (at least 30 min).</li>
		<li>Pre-warm progesterone to room temperature.</li>
		<li>Mix 450 &#181;L of OH-TAM and 225 &#181;L of progesterone in a microcentrifuge tube. Vortex.</li>
		<li>Sonicate at least 10 min and load on to a 1 mL syringe with a 25G needle.</li>
		<li>In the animal facility, weigh the pregnant female (Csf1rMeriCreMer females are in an inbred FVB genetic background and typical weigh between 25 and 33 g).</li>
		<li>Perform an intra-peritoneal injection by slowly injecting the calculated volume into the mouse. After withdrawing the needle, gently press on the puncture wound and massage the abdomen to distribute the OH-TAM. 
		NOTE: The OH-TAM injection dose is 75 mg/kg and the Progesterone dose is 37.5 mg/kg. Table 1 provides the volume to inject into pregnant females.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mouse weight (g)</td>
			<td>Total volume to inject from the mix (&#181;L)</td>
		</tr>
		<tr>
			<td>25</td>
			<td>281</td>
		</tr>
		<tr>
			<td>26</td>
			<td>292</td>
		</tr>
		<tr>
			<td>27</td>
			<td>303</td>
		</tr>
		<tr>
			<td>28</td>
			<td>315</td>
		</tr>
		<tr>
			<td>29</td>
			<td>326</td>
		</tr>
		<tr>
			<td>30</td>
			<td>338</td>
		</tr>
		<tr>
			<td>31</td>
			<td>348</td>
		</tr>
		<tr>
			<td>32</td>
			<td>360</td>
		</tr>
		<tr>
			<td>33</td>
			<td>371</td>
		</tr>
		<tr>
			<td>34</td>
			<td>382</td>
		</tr>
		<tr>
			<td>35</td>
			<td>394</td>
		</tr>
	</tbody>
</table>
Table 1: Injection volume of 4-OH-tamoxifen (OH-TAM). Volume required for a single injection at E8.5 of 75 &#181;g per g (body weight) of OH-TAM supplemented with 37.5 &#181;g per g of progesterone.
2. Dissection of the Yolk Sac (YS) and Fetal Liver (FL)
NOTE: Rigorous sterile techniques are not necessary when manipulating embryos unless if they are going to be used for long-term culture. Nevertheless, the working area must be clean and covered in foil under absorbent paper.
<ol>
	<li>Prepare ice-cold phosphate buffered saline (PBS) and digestion mix (PBS containing 1 mg/mL collagenase D, 100 U/mL Deoxyribonuclease I (DNase I) and 3% fetal bovine serum).</li>
	<li>Sacrifice pregnant females by cervical dislocation at the required gestation day (e.g. embryonic stage E10.5).</li>
	<li>Pinch the skin just over the genitals and make a small incision at the midline with scissors. Pull the skin toward the head to expose completely the body wall without fur. Cut the abdominal muscles to expose the internal organs and push the gut to expose the two uterine horns.</li>
	<li>With middle-sized blunt forceps, grab the fat-pad attached to the ovary and gently pull the uterus. Cut at the cervical level of the uterine horns and lift the horns from the peritoneal cavity. Remove the fat-pad to completely free the uterine horns and cut the horns from the ovary on each side.</li>
	<li>Put the horns into ice-cold PBS in a 10 mm Petri dish. Rapidly grip the uterus muscle layers at one extremity (cervical end) and slide fine scissors between the muscle layer and the decidual tissue to release the embryos with the surrounding decidual tissue. 
	NOTE: Muscle tend to rapidly contract after the extraction, so this step must be as quick as possible.</li>
	<li>Use one pair of fine forceps to cut off the Reichert&#39;s membrane and the placenta.</li>
	<li>Gently remove the yolk sac and place it in a 24-well tissue culture plate with 0.5 mL of digestion mix. 
	NOTE: At this step, embryonic blood can be collected.
	<ol>
		<li>Immediately after severing the umbilical and vitelline vessels, transfer the embryo into a 12-well tissue culture plate containing 10 mM ice-cold ethylenediaminetetraacetic acid (EDTA). Decapitate the embryo using sharp fine scissors, trying to limit as much as possible tissue dilaceration. Incubate on ice for 10-15 min and collect the EDTA containing the blood.</li>
	</ol>
	</li>
	<li>Remove the amnion surrounding the embryo.</li>
	<li>For stages &#60;E11.5, count the number of somite pairs for better staging of embryos and a better time resolution (each somite pair develops in ~1 h 30 min).</li>
	<li>Cut the head of the embryo using forceps or fine scissors. Transfer the head to a 24-well plate with 0.5 mL digestion mix. 
	NOTE: The neuroectoderm and brain at later stages are further dissected for flow cytometry analysis; carefully remove the surrounding vascular plexus.</li>
	<li>Cut the embryo above the hindlimb and remove the forelimbs.</li>
	<li>To isolate the liver, open the thorax using a pair of fine forceps. Pinch anteriorly to the heart and gently pull while using the second forceps to free the organs from the body.
	<ol>
		<li>Carefully separate the fetal liver from the heart and gut. Transfer the fetal liver to a 24-well plate with 0.5 mL digestion mix. Carefully monitor the transfer under the dissecting microscope.</li>
	</ol>
	</li>
	<li>Collect the tail region (or any other embryo part) for genotyping by polymerase chain reaction assay (PCR). 
	NOTE: Other tissues and organs can be harvested from E10.5 embryos for a similar analysis using flow cytometry. Blood, head skin, aorta-gonad-mesonephros region (AGM), heart and neuroectoderm can be collected at E10.5. At later stages, lung, kidney, spleen and pancreas can also be collected. A detailed description of the dissection of the AGM at different developmental stages has been previously described 14.</li>
</ol>
3. Processing of Embryonic Tissues for Flow Cytometry
<ol>
	<li>Incubate the organs (placed in the digestion mix) for 30 min at 37 &#176;C.</li>
	<li>Transfer the tissue and enzymatic solution onto a 100 &#956;m strainer placed in a 6-well tissue culture plate filled with 6 ml of FACS buffer (0.5% Bovine Serum Albumin (BSA) and 2 mM EDTA in 1x PBS). Mechanically dissociate by gently mashing with the black rubber piston of a 2 mL syringe to obtain a single-cell suspension. 
	NOTE: From now on, all steps should be performed at 4 &#176;C.</li>
	<li>Collect the cell suspension with a Pasteur pipette and transfer into a 15 mL tube.</li>
	<li>Spin for 7 min at 320 x g at 4 &#176;C. Discard the supernatant by aspiration.</li>
	<li>Resuspend the pellet in 60 &#181;L Fc-blocking buffer (CD16/CD32 blocking antibody diluted 1/50 in FACS buffer).
	<ol>
		<li>Transfer 50 &#181;L of the single-cell suspension per well in a round bottom 96 multi-well plate. Incubate for at least 15 min on ice. 
		NOTE: The staining can also be performed in 5 mL polystyrene FACS tubes when handling a small number of samples.</li>
		<li>Transfer the remaining 10 &#181;L into a 5 mL polystyrene FACS tube to obtain a pool of the samples from each tissue. This pool will serve as the controls (i.e. unstained samples and fluorescent minus one (FMO) controls for each fluorochrome). Transfer 50 &#181;L of the pool for each fluorescent minus one (FMO) control (one per fluorochrome) to the 96 multi-well plate. 
		NOTE: Each FMO control contains all the fluorochrome-coupled antibodies from the antibody panel, except for the one that is being measured. FMOs are used to identify gate boundaries and to control for the spectral overlap in multicolor panels. To correctly address the variations in background and autofluorescence between different tissues, it is important to prepare these controls from the pool of samples of each tissue. The appropriate control for YFP expression will be the samples from Cre-negative embryos, which are confirmed by PCR genotyping.</li>
	</ol>
	</li>
</ol>
4. Surface Antigen Staining
<ol>
	<li>Prepare the antibody mix in FACS buffer (Table 2). Prepare 50 &#181;L of antibody mix per sample.
	<ol>
		<li>Use the following fluorochrome-coupled antibodies: anti-CD45.2 (clone 104); anti-CD11b (clone M1/70); anti-F4/80 (clone BM8); anti-AA4.1 (clone AA4.1); anti-Kit (clone 2B8); and anti-Ter119 (clone Ter119).</li>
		<li>Prepare six antibody mixes for the FMO controls in FACS buffer. Prepare 50 &#181;L of antibody mix per control sample. 
		NOTE: The antibody dilution in the antibody mix is two times more concentrated than the final concentration indicated in the materials table. For a panel with six fluorochrome-coupled antibodies, 6 FMO controls are prepared. Table 2 provides the composition of the antibody mix and FMO controls for one tube.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td colspan="7">Volume (&#181;L) of antibody&#160; (Final Volume 50 &#181;L)</td>
		</tr>
		<tr>
			<td>Antibody</td>
			<td>Clone</td>
			<td>Antibody Mix</td>
			<td>FMO CD45.2</td>
			<td>FMO CD11b</td>
			<td>FMO F4/80</td>
			<td>FMO AA4.1</td>
			<td>FMO&#160;&#160;&#160;&#160;&#160; Kit</td>
			<td>FMO Ter119</td>
		</tr>
		<tr>
			<td>anti-CD45.2</td>
			<td>104</td>
			<td>1</td>
			<td>0</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>anti-CD11b</td>
			<td>&#160;M1/70</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>anti-F4/80</td>
			<td>BM8</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>0</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>anti-AA4.1</td>
			<td>AA4.1</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>0</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>anti-Kit</td>
			<td>2B8</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>anti-Ter119</td>
			<td>Ter119</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0</td>
		</tr>
		<tr>
			<td colspan="2">FACS buffer</td>
			<td>45.5</td>
			<td>46.5</td>
			<td>46</td>
			<td>46.5</td>
			<td>46.5</td>
			<td>46</td>
			<td>46</td>
		</tr>
	</tbody>
</table>
Table 2: Volume of antibodies using for staining and Fluorescent minus one (FMO) controls. Volume (&#181;L) of antibody required for a final volume of 50 &#181;L.
<ol start="2">
	<li>Add 50 &#181;L of the antibody mix to the samples in the 96-multiwell plate (final volume during staining is 100 &#181;L). Gently pipette up and down twice and incubate on ice for 30 min.</li>
	<li>Spin the 96-multiwell plate for 7 min at 320 x g at 4 &#176;C and discard the supernatant. Resuspend in 200 &#181;L of FACS buffer.</li>
	<li>Repeat wash procedure (step 4.3) twice with 150 &#181;L FACS buffer.</li>
	<li>Filter the samples and the controls (FMOs and unstained pool samples) into 5 ml polystyrene tubes through a 70 &#956;m strainer. Store on ice until acquisition.</li>
</ol>
5. Identification of EMPs and YS-derived Macrophages by Flow Cytometry
NOTE: This protocol was optimized using a 4 laser flow cytometer equipped with a 405 nm Violet laser, a 488 nm Blue laser, a 562 nm Yellow laser and a 638 nm Red laser.
<ol>
	<li>Prepare compensation beads for each coupled antibody following the manufacturer&#39;s instructions. Use unstained samples and compensation beads to optimize laser intensities.</li>
	<li>To identify gate boundaries, use FMO (Fluorescence minus one) controls for each antibody.</li>
	<li>In order to exclude dead cells, add 1 &#181;L of DAPI (1 mg/ml) to the tube (containing 200 &#181;L of stained cell suspension) 1 min before acquisition. Use the strong DAPI signal and the forward-scattered parameter (FSC) to perform exclusion of dead cells and debris. 
	NOTE: Use software from the flow cytometer to draw gates during acquisition. Compensation matrix and analysis can be performed after sample acquisition using other commercially available software.</li>
	<li>Use forward and side scatter (FSC vs SSC) to perform live cell and doublet discrimination from total events.</li>
	<li>Create fluorescence dot plots in log-scale axis and draw daughter gates to, first, exclude erythrocytes (Ter119+) and, then, to identify progenitor cells (Kit+) and hematopoietic cells (CD45+ Kitneg) (See Figure 1).</li>
	<li>Create fluorescence histograms to quantify the labeling efficiency of YFP among the different identified populations in the YS (Figure 2), fetal liver (Figure 3) and brain (Figure 4).</li>
</ol>

<ol>
	<li>Bertrand, J. 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=Three+pathways+to+mature+macrophages+in+the+early+mouse+yolk+sac.">Three pathways to mature macrophages in the early mouse yolk sac.</a> Blood. 106 (9), 3004-3011 (2005).</li><li>Palis, J., Robertson, S., Kennedy, M., Wall, C., Keller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+erythroid+and+myeloid+progenitors+in+the+yolk+sac+and+embryo+proper+of+the+mouse.">Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse.</a> Development. 126 (22), 5073-5084 (1999).</li><li>Chen, M. 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=Erythroid/myeloid+progenitors+and+hematopoietic+stem+cells+originate+from+distinct+populations+of+endothelial+cells.">Erythroid/myeloid progenitors and hematopoietic stem cells originate from distinct populations of endothelial cells.</a> Cell Stem Cell. 9 (6), 541-552 (2011).</li><li>Frame, J. M., Fegan, K. H., Conway, S. J., McGrath, K. E., Palis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definitive+Hematopoiesis+in+the+Yolk+Sac+Emerges+from+Wnt-Responsive+Hemogenic+Endothelium+Independently+of+Circulation+and+Arterial+Identity.">Definitive Hematopoiesis in the Yolk Sac Emerges from Wnt-Responsive Hemogenic Endothelium Independently of Circulation and Arterial Identity.</a> Stem Cells. , (2015).</li><li>Kieusseian, A., Brunetdela de la Grange, P., Burlen-Defranoux, O., Godin, I., Cumano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immature+hematopoietic+stem+cells+undergo+maturation+in+the+fetal+liver.">Immature hematopoietic stem cells undergo maturation in the fetal liver.</a> Development. 139 (19), 3521-3530 (2012).</li><li>Gomez Perdiguero, ., E, , 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=Tissue-resident+macrophages+originate+from+yolk-sac-derived+erythro-myeloid+progenitors.">Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors.</a> Nature. 518 (7540), 547-551 (2015).</li><li>Hoeffel, G., 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=C-Myb(+)+erythro-myeloid+progenitor-derived+fetal+monocytes+give+rise+to+adult+tissue-resident+macrophages.">C-Myb(+) erythro-myeloid progenitor-derived fetal monocytes give rise to adult tissue-resident macrophages.</a> Immunity. 42 (4), 665-678 (2015).</li><li>Kierdorf, K., Prinz, M., Geissmann, F., Gomez Perdiguero, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+function+of+tissue+resident+macrophages+in+mice.">Development and function of tissue resident macrophages in mice.</a> Semin Immunol. 27 (6), 369-378 (2015).</li><li>Houssaint, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+the+mouse+hepatic+primordium.+II.+Extrinsic+origin+of+the+haemopoietic+cell+line.">Differentiation of the mouse hepatic primordium. II. Extrinsic origin of the haemopoietic cell line.</a> Cell Differ. 10 (5), 243-252 (1981).</li><li>Cumano, A., Godin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+the+hematopoietic+system.">Ontogeny of the hematopoietic system.</a> Annu Rev Immunol. 25, 745-785 (2007).</li><li>McGrath, K. E., 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=Distinct+Sources+of+Hematopoietic+Progenitors+Emerge+before+HSCs+and+Provide+Functional+Blood+Cells+in+the+Mammalian+Embryo.">Distinct Sources of Hematopoietic Progenitors Emerge before HSCs and Provide Functional Blood Cells in the Mammalian Embryo.</a> Cell Rep. 11 (12), 1892-1904 (2015).</li><li>Schulz, 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=A+lineage+of+myeloid+cells+independent+of+Myb+and+hematopoietic+stem+cells.">A lineage of myeloid cells independent of Myb and hematopoietic stem cells.</a> Science. 336 (6077), 86-90 (2012).</li><li>Chevalier, C., Nicolas, J. F., Petit, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+delivery+of+4-hydroxy-tamoxifen+for+clonal+and+polyclonal+labeling+of+cells+of+the+surface+ectoderm,+skin,+and+hair+follicle.">Preparation and delivery of 4-hydroxy-tamoxifen for clonal and polyclonal labeling of cells of the surface ectoderm, skin, and hair follicle.</a> Methods Mol Biol. 1195, 239-245 (2014).</li><li>Bertrand, J. Y., Giroux, S., Cumano, A., Godin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cell+development+during+mouse+embryogenesis.">Hematopoietic stem cell development during mouse embryogenesis.</a> Methods Mol Med. 105, 273-288 (2005).</li><li>Bertrand, J. 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=Characterization+of+purified+intraembryonic+hematopoietic+stem+cells+as+a+tool+to+define+their+site+of+origin.">Characterization of purified intraembryonic hematopoietic stem cells as a tool to define their site of origin.</a> Proc Natl Acad Sci U S A. 102 (1), 134-139 (2005).</li><li>Ginhoux, F., 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=Fate+mapping+analysis+reveals+that+adult+microglia+derive+from+primitive+macrophages.">Fate mapping analysis reveals that adult microglia derive from primitive macrophages.</a> Science. 330 (6005), 841-845 (2010).</li><li>Nakamura, E., Nguyen, M. T., Mackem, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+tamoxifen-regulated+Cre+activity+in+mice+using+a+cartilage-specific+CreER(T)+to+assay+temporal+activity+windows+along+the+proximodistal+limb+skeleton.">Kinetics of tamoxifen-regulated Cre activity in mice using a cartilage-specific CreER(T) to assay temporal activity windows along the proximodistal limb skeleton.</a> Dev Dyn. 235 (9), 2603-2612 (2006).</li><li>Qian, B. Z., 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=CCL2+recruits+inflammatory+monocytes+to+facilitate+breast-tumour+metastasis.">CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis.</a> Nature. 475 (7355), 222-225 (2011).</li><li>Yona, S., 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=Fate+mapping+reveals+origins+and+dynamics+of+monocytes+and+tissue+macrophages+under+homeostasis.">Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis.</a> Immunity. 38 (1), 79-91 (2013).</li><li>Sheng, J., Ruedl, C., Karjalainen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Most+Tissue-Resident+Macrophages+Except+Microglia+Are+Derived+from+Fetal+Hematopoietic+Stem+Cells.">Most Tissue-Resident Macrophages Except Microglia Are Derived from Fetal Hematopoietic Stem Cells.</a> Immunity. 43 (2), 382-393 (2015).</li></ol>

The authors have nothing to disclose.

The different waves of hematopoietic precursor cells partially overlap within a short time frame, which makes the analysis of the contribution of each wave of developmental hematopoiesis to immune cells technically very challenging.
Tamoxifen-inducible Cre systems offer the opportunity to tag specific cells in a temporally inducible manner and to perform lineage analysis in embryos or adults, without the need for ex vivo or in vitro culture or transplantation. In tamoxifen-inducible Cre strains, a fusion gene is created between a bacterial Cre recombinase and a mutant form of the ligand binding domain of the human estrogen receptor (CRE-ER) or between an improved single-point mutant Cre and two mouse estrogen receptors (Mer-iCre-Mer). Fusion of Cre with estrogen receptors leads to the cytoplasmic sequestration of Cre by Hsp90, thereby preventing nuclear Cre-mediated recombination. Tamoxifen (TAM) is metabolized by the liver into 4-hydroxytamoxifen (OH-TAM), its active metabolite with high binding affinity for estrogen receptor. OH-TAM binding to the fusion Cre leads to the disruption of the interaction with Hsp90, permitting its translocation to the nucleus and initiation of Cre-mediated recombination. Injection of TAM or OH-TAM in utero into pregnant females has been shown to activate recombination in the developing embryo. Tamoxifen administration during pregnancy can lead to abortion and thus reduces litter size but it also prevents birth if delivered after mid-gestation, in which case pup delivery through a caesarean operation is required. To counterbalance tamoxifen effects during pregnancy, we supplement OH-TAM administration with a half-dose of progesterone, in order to improve survival of embryos and reduce the risk of abortions 17.
Several routes can be employed to deliver TAM or OH-TAM into pregnant females. Currently oral gavage or intra peritoneal (ip) injection is used for tamoxifen. OH-TAM has the advantage to be immediately available, as it does not require to be metabolized by the liver of the pregnant dam. However, while tamoxifen is very easy to dissolve in corn oil, OH-TAM is very difficult to prepare using the same technique and results in an emulsion. This has been a limiting step in employing OH-TAM for in utero pulse labeling. We have adapted the protocol for preparation of OH-TAM developed by the group of J.F. Nicolas 13, where they use PEG-35 castor oil, a solvent with amphiphilic properties to solubilize OH-TAM, as tamoxifen dissolution is one of the most critical steps in the procedure. This allows for reproducible and optimal preparation of OH-TAM and considerably shortens the time of tamoxifen preparation. Further, it is necessary to adapt the concentration of OH-TAM to inject when using different inducible Cre strains. The combination of a viability dye (DAPI) and size (FSC) is critical to remove dead cells and cellular debris, respectively. Dead cells and debris are highly autofluorescent in most of the violet, blue and red laser detectors, and can hamper flow cytometric analysis. Further, we do not recommend fixing the cells after staining prior to analysis with the flow cytometer. Fixation can lead to changes in cell morphology and thus renders difficult size discrimination based on Forward and Side Scatter (FSC vs. SSC). Further, fixation of samples with PFA leads to a significant loss of YFP fluorescent intensity.
The main limitation within this protocol is that the populations are not tested on their functionality and differentiation potential. In order to overcome this, cells can be sorted following a similar protocol using a Fluorescently-activated cell sorter (FACS) to perform colony-forming assay. In this case, the procedure must be performed under sterile conditions and use of fetal bovine serum (FBS) in the FACS buffer, instead of BSA, is highly recommended. The low labeling efficiency achieved with this technique and the consequently low number of YFP+ cells of interest per embryonic tissue is a major challenge in the study of EMP phenotype. This protocol uses a 4-laser flow cytometer. A flow cytometer with 3 lasers can also be used but it is important to adapt the antibody panel to take into account the increased spectral overlap between dyes. Additionally, titration of antibodies is required to find the appropriate concentration when changing the antibody panel and we recommend performing titration of antibodies and validation of the new antibody panel in a pilot experiment where all embryos per tissue are pooled, in order to increase the number of cells analyzed per tissue.
The field of developmental biology has been revolutionized by the Cre/Lox method, which allows permanent labeling of cells in situ. This approach achieves tissue- or cell type -specificity through expression of the Cre recombinase under the control of a specific promoter. In our case, we chose to study the expression of the Cre recombinase under the control of the promoter of Csf1r (Colony Stimulating Factor 1 Receptor), an important mitogenic and growth factor receptor for macrophages and their progenitors, using a strain developed by the group of J. Pollard 18. The advantage of a fate mapping strategy based on Csf1r expression compared to other published strategies is that it allows the labeling of yolk sac-derived cells (EMP and macrophages) without labeling any fetal HSCs 12. The Cx3cr1CreERT2 strain can also label yolk sac cells without labeling HSCs 19, however it can only label macrophages and macrophage precursors but it does not label EMP progenitors 1. The Cx3cr1CreERT2 strain has the same caveat as the Csf1rMerCreMer line, since it cannot distinguish between macrophages derived from unipotent primitive macrophage progenitors or from EMP. When tamoxifen is administered as early as E7.5 in both Runx1MerCreMer16 and KitMerCreMer20, peripheral blood cells are always labeled in adults, albeit at a very low efficiency, thus demonstrating labeling of HSCs during development. Further, KitMerCreMer fate mapping labels yolk sac Kit+ Sca1+ progenitors while EMP are Kit+ Sca1 negative 11. The Csf1rMeriCreMer strain is not a knock-in but it was generated by classical additive transgenesis, thus expression of the Cre may not fully recapitulate endogenous expression of Csf1r. However, this has the advantage to avoid any potential developmental defects due heterozygous expression of Csf1r. This is an important factor to take to account when analyzing other fate mapping strategies used for developmental hematopoiesis studies, such as Runx1MerCreMer, KitMerCreMer and Cx3cr1CreERT2, where the inducible Cre is inserted-in the endogenous locus. For both Runx1MerCreMer and Kit MerCreMer, developmental hematopoietic defects have been described in heterozygous embryos. Collectively, the method presented here allows to investigate yolk sac EMP biology during development and also yolk sac-derived macrophages found in adult tissues and distinct from HSC-derived macrophages. This will improve our current understanding of this lineage of resident macrophages and their specific functions in adulthood. The immunophenotypic characterization of EMPs has been improved recently using cell sorting and colony forming assays, demonstrating that yolk sac EMP specifically express CD41 and CD16/32 in early stages of development 11. However, the specificity of expression of CD41 and CD16/32 needs further characterization from E10.5 onwards, especially in the fetal liver niche, using fate-mapping models. Indeed, whether CD41 and CD16/32 expression is still specific to EMP when compared to fetal HSCs and HSC-derived progenitors in the E10.5 to E14.5 fetal liver need to be elucidated. The presented method allows to label EMP generated in the yolk sac and to follow them during embryonic development and it will contribute to a more detailed characterization of EMP phenotype when they seed the fetal liver.
This method could be important in the near future to study EMP differentiation pathways. While EMPs emerge from the yolk sac endothelium from E8.5 to E10.5 4, this method only allows to label a fraction of the progenitors emerging between E8.5-E9.5. Therefore, a limitation of this method is that only a small fraction of EMP is labeled, thus rendering difficult the analysis in classical loss-of-function studies with floxed alleles for candidate genes. Alternatively, sparse labeling could become an advantage in clonal studies of EMP differentiation in vivo, using clonal reporter. Nevertheless, the labeling efficiency needs to be characterized for each floxed reporter or floxed allele used, as recombination efficiency of floxed constructs depends on genomic locus (floxed alleles) or the Rosa26 reporter construct. Indeed, different Rosa26 reporter lines are available with different promoters and it is reported that Rosa26tdTomato and Rosa26mTmG strain have higher recombination efficiency than the Rosa26LSL-eYFP line. The reporter mouse strain used in this protocol is Rosa26LSL-eYFP line, which cannot provide information regarding the dynamic process of progenitor maturation mentioned in the results section. The question of maturation process can be partially addressed using different reporter strains like the Rosa26mTmG. In such strain, cells can be distinguished based on the time elapsed since the recombination event. All cells in the Rosa26mTmG strain express membrane-bound tdTomato fluorescent protein and Cre recombination excises the tdTomato cassette and allows for expression of membrane-bound GFP. Since tdTomato has a long half-life, cells that still contain tdTomato but have started to express the GFP (shortly after recombination) are tdTomato+ GFPlow/int and can be distinguished from tdTomatoneg GFP+ cells that do not express tdTomato anymore and express higher levels of GFP (later after recombination).
As discussed above, OH-TAM preparation is a critical step to consistently deliver similar doses of OH-TAM between experiments and to reduce tamoxifen side effects. Progesterone co-administration is also useful to limit deleterious effect of tamoxifen on pregnancy. Another critical element of the protocol is the viability of the single-cell suspension obtained from different embryonic tissues. We usually obtain &#62;90% viable cells for flow cytometric analysis. To achieve this, it is critical to follow all the steps rapidly and to maintain all samples on ice after tissue collection. Appropriate controls such as unstained samples, single stains and fluorescent minus one (FMO) controls are required to identify gate boundaries and to control for the spectral overlap in multicolor panels. Changes in the antibody need to be validated in terms of antibody titration and spectral overlap. Finally, the choice of the Rosa26 reporter strain needs to be adapted to the scientific question that is investigated.

There are several successive but overlapping waves of hematopoietic progenitors during development whose myeloid progeny remain into adulthood. First, unipotent &#34;primitive&#34; progenitors emerge in the mouse yolk sac 1,2 between E7.5-E8.25 and give rise to embryonic macrophages without any monocytic intermediate. Whether macrophages derived from primitive progenitors persist in the adult brain as microglia remains a subject of active investigation. Second, Erythro-Myeloid Precursors (EMPs) arise in the yolk sac at E8.5, enter the bloodstream and colonize the embryo. EMPs emerge from the yolk sac hemogenic endothelium in a Runx1-dependant endothelial-to-hematopoietic transition 3,4. While EMP can differentiate into macrophages within the yolk sac, they also colonize the fetal liver from embryonic day (E)95 and differentiate into erythrocytes, megakaryocytes, macrophages, monocytes granulocytes and mast cells 6. The macrophages that derive from EMPs exhibit proliferative capacity in developmental and adult tissues. Whether EMP-derived macrophages bypass the monocyte stage of differentiation is still controversial as very little is known about their differentiation pathway 7,8. Finally, Hematopoietic Stem Cells (HSCs) emerge at E10.5 within the embryo proper from the aorta-gonad-mesonephros region and migrate to the fetal liver. HSCs with long-term repopulation capacity are only detected after E11 (at the 42 somite pair stage)9. There, they expand and differentiate from E12.5 until definitive hematopoiesis begins to shift to the bone marrow, which becomes the predominant site of blood cell production for the duration of postnatal life 10.
The spatial and temporal overlap in emergence, as well as shared immuno-phenotypic markers has thus far hampered our ability to distinguish the specific contributions of these waves of embryonic hematopoietic progenitors. While both EMP and HSC are generated in a Runx1-dependent manner and express the transcription factor Myb and the growth factor receptor Csf1r (Colony-Stimulating Factor 1 Receptor, also known as Macrophage Colony Stimulating Factor Receptor) among others, EMPs can be distinguished from HSCs by their lack of lymphoid potential, both in vitro and in vivo, their lack of long-term repopulating potential and lack of surface expression of the lineage marker Sca-1 11. Genetic fate mapping models are required to characterize macrophage ontogeny since they allow targeting embryonic progenitors in a cell-specific and time-specific manner. Here we present the fate-mapping protocol used in our laboratory to discriminate between the two lineages of macrophages found in most adult tissues: HSC-derived infiltrating macrophages and HSC-independent resident macrophages.
Tissue resident macrophages have been traced back to Myb-independent precursor cells expressing the cytokine receptor Csf1r12 and are present in the embryo at E8.5-E10.5 using three complementary fate-mapping strategies 6. In order to study yolk sac hematopoiesis without labeling fetal HSCs, we use a transgenic strain, Csf1rMeriCreMer, expressing a tamoxifen-inducible fusion protein of &#39;improved&#39; Cre recombinase and two mouse estrogen receptors (Mer-iCre-Mer) under the control of the Csf1r promoter. Hence, the Cre recombinase will be active in Csf1r-expressing cells during a limited time-window. When used with a reporter strain containing a fluorescent protein downstream of a lox-STOP-lox cassette (Rosa26LSL-eYFP), it will lead to the permanent genetic labeling of the cells present at the time of induction but also of their progeny. Administration at E8.5 of the active form of tamoxifen, 4-hydroxytamoxifen (OH-TAM), labels EMPs and macrophages, without labeling yolk sac unipotent &#34;primitive&#34; progenitors or fetal HSCs. Thereby, we have characterized the immunophenotype of EMPs and their progeny during embryonic development, as well as assessed the contribution of yolk sac-derived macrophages to the adult macrophage pools. Further work is required to characterize whether primitive progenitor-derived macrophages are also labeled using this approach and whether they can contribute to adult macrophage pools.

<table><tbody><tr><td>#5 Straight Forceps</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>MC19/B Pascheff-Wolff Spring Scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>15371-92</td><td></td></tr><tr><td>8.5 cm straight scissors</td><td>Fine Science Tools</td><td>14090-09</td><td></td></tr><tr><td>Anti-Mouse Kit-PE (clone 2B8)</td><td>BD Pharmingen</td><td>553355</td><td>1/200 dilution in FACs Buffer</td></tr><tr><td>Anti-Mouse CD45.2 APC-Cy7 (clone 104)</td><td>Sony</td><td>1149120</td><td>1/100 dilution in FACs Buffer</td></tr><tr><td>Anti-Mouse AA4.1-APC (CD93, clone AA4.1)</td><td>eBioscience</td><td>17-5892</td><td>1/100 dilution in FACs Buffer</td></tr><tr><td>Anti-Mouse Ter119 PerCP-Cy5.5 (clone Ter119)</td><td>Biolegend</td><td>560512</td><td>1/200 dilution in FACs Buffer</td></tr><tr><td>Anti-Mouse F4/80-BV421 (clone BM8)</td><td>BD Pharmingen</td><td>123137</td><td>1/100 dilution in FACs Buffer</td></tr><tr><td>Anti-Mouse CD11b PE-Cy7 (clone M1/70)</td><td>BD Pharmingen</td><td>552850</td><td>1/200 dilution in FACs Buffer</td></tr><tr><td>Anti-Mouse CD16/CD32 (Mouse BD Fc Block, clone 2.4G2)</td><td>BD Biosciences</td><td>553142</td><td></td></tr><tr><td>PBS 1x</td><td>Fischer Scientific</td><td>12559069</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Fischer Scientific</td><td>11570506</td><td></td></tr><tr><td>Collagenase D&amp;nbsp;</td><td>Roche</td><td>11088882001</td><td></td></tr><tr><td>Deoxyribonuclease I</td><td>Sigma</td><td>D4527-20KU</td><td></td></tr><tr><td>6-well tissue culture plate</td><td>Dutscher Dominique</td><td>353046</td><td></td></tr><tr><td>12-well tissue culture plate</td><td>Dutscher Dominique</td><td>353224</td><td></td></tr><tr><td>24-well tissue culture plate</td><td>Fischer Scientific</td><td>11874235</td><td></td></tr><tr><td>96 wells U-shape bottom tissue culture plate</td><td>Dutscher Dominique</td><td>353227</td><td></td></tr><tr><td>&amp;nbsp;Syringe Plastipak 2 mL&amp;nbsp;</td><td>Dutscher Dominique</td><td>300185</td><td></td></tr><tr><td>Nylon grid 100 &amp;micro;m cell strainers</td><td>Dutscher Dominique</td><td>352360</td><td></td></tr><tr><td>Nylon grid 70 &amp;micro;m cell strainers</td><td>Dutscher Dominique</td><td>352350</td><td></td></tr><tr><td>15 ml Falcon tubes</td><td>Dutscher Dominique</td><td>352096</td><td></td></tr><tr><td>Stericup GP Millipore filtration kit, 0.2 &amp;mu;m</td><td>Dutscher Dominique</td><td>51246</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A7906-500G</td><td></td></tr><tr><td>(Z)-4-HYDROXYTAMOXIFEN 25mg</td><td>Sigma</td><td>H7904-25MG</td><td>Special care should be taken when preparing and working with tamoxifen and its derivates. Always use appropriate personal protective equipment</td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P3972</td><td>Always use appropriate personal protective equipment</td></tr><tr><td>PEG-35 castor oil (Kolliphor/Cremophor EL)</td><td>Sigma</td><td>C5135</td><td></td></tr><tr><td>Sunflower oil</td><td>Sigma</td><td>S5007-250ML</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Sigma</td><td>24103-1L-R-D</td><td>Always use appropriate personal protective equipment and use under the fumehood</td></tr><tr><td>EDTA</td><td>Sigma</td><td>E9884-500G</td><td></td></tr><tr><td>DAPI, 1 ML (1 MG/ML IN WATER)</td><td>Fischer Scientific</td><td>10116287</td><td></td></tr><tr><td>CytoFLEX S B2-R3-V4-Y4 flow cytometer</td><td>Beckman Coulter</td><td>B75408</td><td></td></tr><tr><td>CytoFLEX Daily QC Fluorospheres</td><td>Beckman Coulter&amp;nbsp;</td><td>B53230</td><td></td></tr><tr><td>VersaComp Antibody Capture Bead kit (2x5 mL)</td><td>Beckman Coulter&amp;nbsp;</td><td>B22804</td><td></td></tr><tr><td>FVB-Tg(Csf1r-cre/Esr1*)1Jwp/J</td><td>The Jackson laboratory</td><td>19098</td><td></td></tr><tr><td>B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J</td><td>The Jackson laboratory</td><td>6148</td><td></td></tr></tbody></table>

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.

Genetic fate mapping was achieved by administration at E8.5 of OH-TAM into Csf1r-Mer-iCre-Mer females mated with males carrying a Rosa26-LSL-eYFP reporter. In the presence of OH-TAM, excision of the stop cassette leads to the permanent expression of YFP in the cells expressing Csf1r. We collected two hematopoietic tissues: the yolk sac and the fetal liver and a non-hematopoietic tissue, the neuroectoderm, from the embryos at E10.5. Single-cell suspensions were obtained by enzymatical and mechanical dissociation and samples were analyzed by flow cytometry after staining with fluorescent-coupled antibodies. After exclusion of dead cells and doublets, single cell suspensions were analyzed (Figure 1). All gates were defined using Fluorescence Minus One (FMO) controls. It is recommended to also perform an erythrocyte exclusion (Ter119+ cells) to improve clarity of the analysis (Figure 1B). Using the cell surface markers Kit (progenitor marker) and CD45 (hematopoietic cells marker), we were able to distinguish three populations of cells of interest: Kit+ CD45neg; Kit+ CD45low and Kitneg CD45+ (Figure 1). Progenitors were further analyzed based on their expression of AA4.1 (Figures 2-4). Indeed, AA4.1 is a surface marker that allows the enrichment of the progenitor population within erythromyeloid progenitors in the yolk sac, as demonstrated in colony forming assays 15. Once the populations of interest were identified, we quantified the YFP labeling efficiency in each population using histograms. Among the yolk sac Kit+ progenitors, both CD45neg (Figure 2A) and CD45low (Figure 2B) cell populations contain AA4.1+ YFP+ cells. However, AA4.1+ YFP+ cells in the liver and brain are only found in the Kit+ CD45low progenitor population (Figure 3B and 4B). Since, the brain is not an active site of hematopoiesis, progenitors found in this tissue could correspond to circulating progenitors. This also suggests a process of progenitor maturation as they migrate from the yolk sac to the fetal liver and the peripheral tissues. Kit+ progenitors first express AA4.1, irrespectively of CD45 expression, but by the time they reach circulation and the fetal liver, all AA4.1+ YFP+ progenitors express detectable levels of cell surface CD45. Macrophages, defined as F4/80bright CD11b+, were found in the Kitneg CD45+ gate (Figure 2-4). Macrophages in the E10.5 yolk sac, liver and brain were efficiently labeled (60 to 80% of F4/80bright CD11b+ cells are YFP+) by a single OH-TAM administration at E8.5 (Figure 2C, 3C and 4C).
To compare labeling efficiency between litters, we recommend that an internal control for recombination efficiency is used. Variability in the level of expression of the reporter can be observed between experiments, due to variations in developmental timing between litters and mouse strains when OH-TAM is injected in utero. Hence, we recommend that embryo staging from E8.5 to E11.5 embryos is performed by somite pair counting. In this protocol, we propose to use the labeling efficiency in the brain resident macrophages (microglia) as a reference to compare the efficiency of Cre recombination between different experiments and strains (Figure 4C). Other resident macrophages could be used, however microglia were the first cells to be described as yolk sac-derived macrophages 16 and they represent the most abundant population of tissue resident macrophages at E10.5. Thus, YFP labeling in microglia can be used to assess the fate-mapping efficiency in each experiment and compare data from different litters.
<img alt="Figure 1" src="/files/ftp_upload/55305/55305fig1.jpg" /> 
Figure 1: Gating strategy to identify progenitor cells (Kit+ CD45neg and Kit+ CD45low) and differentiated hematopoietic cells (Kitneg CD45+). (A) Discrimination of live cells and singlets is performed using DAPI staining and Forward and Side scatter (FSC/SSC) parameters. (B) Following red blood cell exclusion (Ter119neg), three populations of cells of interest are identified based on cell surface expression of Kit and CD45: Kit+ CD45neg; Kit+ CD45low and Kitneg CD45+ cells. (C) Csf1r-expressing cells present when OH-TAM is injected and their progeny are identified in Cre-recombinase positive embryos as expressing YFP compared to the Cre-recombinase negative embryos (confirmed later by polymerase chain reaction assay, PCR). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55305/55305fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55305/55305fig2.jpg" /> 
Figure 2: Labeling efficiency in the yolk sac from E10.5 Csf1rMeriCreMer Rosa26LSL-eYFP embryospulsed with OH-TAM at E8.5. (A) Kit+ CD45neg cells are gated based on Kit and CD45 expression on live yolk sac cells (blue gate, left panel). AA4.1 and Kit expression on Kit+ CD45neg cells. Blue gate encloses AA4.1+ Kit+ CD45neg cells (middle panel). Comparison of YFP labeling in AA4.1+ Kit+ CD45neg cells (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). (B) Kit+ CD45low cells are gated based on Kit and CD45 expression (blue gate, left panel). AA4.1 and Kit expression on Kit+ CD45low cells. Blue gate encloses EMP, as defined as AA4.1+ Kit+ CD45low cells (middle panel). Comparison of YFP labeling in AA4.1+ Kit+ CD45low cells (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). (C) Hematopoietic cells are defined as Kitneg CD45+ and gated in blue (left panel). F4/80 and CD11b expression on Kitneg CD45+ cells. Macrophages are defined as F4/80bright CD11b+ cells (middle panel). Comparison of YFP labeling in F4/80bright CD11b+ macrophages (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55305/55305fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55305/55305fig3.jpg" /> 
Figure 3: Labeling efficiency in the liver from E10.5 Csf1rMeriCreMer Rosa26LSL-eYFP embryospulsed with OH-TAM at E8.5. (A) Kit+ CD45neg cells are gated based on Kit and CD45 expression on live liver cells (blue gate, left panel). AA4.1 and Kit expression on Kit+ CD45neg cells. Blue gate encloses AA4.1+ Kit+ CD45neg cells (middle panel). Comparison of YFP labeling in AA4.1+ Kit+ CD45neg cells (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). (B) Kit+ CD45low cells are gated based on Kit and CD45 expression (blue gate, left panel). AA4.1 and Kit expression on Kit+ CD45low cells. Blue gate encloses EMP, as defined as AA4.1+ Kit+ CD45low cells (middle panel). Comparison of YFP labeling in AA4.1+ Kit+ CD45low cells (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). (C) Hematopoietic cells are defined as Kitneg CD45+ and gated in blue (left panel). F4/80 and CD11b expression on Kitneg CD45+ cells. Macrophages are defined as F4/80bright CD11b+ cells (middle panel). Comparison of YFP labeling in F4/80bright CD11b+ macrophages (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55305/55305fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55305/55305fig4.jpg" /> 
Figure 4: Labeling efficiency in the brain from E10.5 Csf1rMeriCreMer Rosa26LSL-eYFP embryospulsed with OH-TAM at E8.5. (A) Kit+ CD45neg cells are gated based on Kit and CD45 expression on live brain cells (blue gate, left panel). AA4.1 and Kit expression on Kit+ CD45neg cells. Blue gate encloses AA4.1+ Kit+ CD45neg cells (middle panel). Comparison of YFP labeling in AA4.1+ Kit+ CD45neg cells (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). (B) Kit+ CD45low cells are gated based on Kit and CD45 expression (blue gate, left panel). AA4.1 and Kit expression on Kit+ CD45low cells. Blue gate encloses EMP, as defined as AA4.1+ Kit+ CD45low cells (middle panel). Comparison of YFP labeling in AA4.1+ Kit+ CD45low cells (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). (C) Hematopoietic cells are defined as Kitneg CD45+ and gated in blue (left panel). F4/80 and CD11b expression on Kitneg CD45+ cells. Macrophages are defined as F4/80bright CD11b+ cells (middle panel). Comparison of YFP labeling in F4/80bright CD11b+ macrophages (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55305/55305fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry at JoVE.com

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo 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 ...

identification-erythromyeloid-progenitors-their-progeny-mouse-embryo

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

12.9K Views.  Institut Pasteur. 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. 

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

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

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

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

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

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

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

Immunology and Infection

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 ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As such, it contains hematopoietic stem and progenitor cells (HSPCs), as well as supporting stromal cells that contribute to the maintenance and regulation of HSPCs. Hematological and other BM disorders disrupt hematopoiesis by affecting hematopoietic cells directly and/or through the alteration of the BM niche. Here, we describe a method to study hematopoiesis in health and malignancy through the phenotypic analysis of murine BM HSPCs and stromal niche populations by flow cytometry. Our method details the required steps to enrich BM cells in endosteal and central BM fractions, as well as the appropriate gating strategies to identify the two key niche cell types involved in HSPC regulation, endothelial cells and mesenchymal stem cells. The phenotypic analysis proposed here may be combined with mouse mutants, disease models, and functional assays to characterize the HSPC compartment and its niche.

Here, we describe a simple protocol for the isolation and staining of murine bone marrow cells to phenotype hemopoietic stem and progenitor cells along with the supporting niche endothelial and mesenchymal stem cells. A method to enrich cells located in endosteal and central bone marrow areas is also included.

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As ...

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

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Chapters in this video

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

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

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

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

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

Flow Cytometric Characterization of Murine B Cell Development

Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells