LM was supported by a grant from the Lady Tata Memorial Trust. JR was supported by a PhD fellowship from Funda&#231;&#227;o para a Ci&#234;ncia e Tecnologia (FCT; FCT fellowship UI/BD/150833/2021). ML was supported by a PhD fellowship from FCT (FCT fellowship 2021.04773.BD). DD was supported by grants from the American Society of Hematology, the Pablove Foundation, FCT (EXPL/MED-ONC/0522/2021), and the Portuguese Society of Hematology. We thank the support from Dr. Catarina Meireles and Emilia Cardoso of TRACY facility at i3s.

The animals used in this protocol were housed at the i3S animal facility under specific pathogen-free conditions in a 12 h light-dark cycle and temperature-controlled environment. Free access to standard rodent chow and water was provided. All the animals received humane care according to the criteria outlined by the Federation of European Laboratory Animal Science Associations for the care and handling of laboratory animals (EU Directive 2010/63/EU). The experimental procedure performed on the animals (euthanasia) was approved by the i3S Animal Ethics Committee (ref. DD_2019_15) and the Dire&#231;&#227;o-Geral de Alimenta&#231;&#227;o e Veterin&#225;ria. Details of the materials used throughout this protocol can be found in the Table of Materials.
1. Preparation of solutions and staining cocktails
<ol>
	<li>Phosphate-buffered saline (PBS): Dissolve five PBS tablets into 1 L of distilled water. Store at room temperature (RT).</li>
	<li>PBS 2% fetal bovine serum (FBS): Add 10 mL of FBS to 500 mL of PBS. Store at 4 &#176;C.</li>
	<li>Red blood cell (RBC) lysis buffer (1x): Add 50 mL of 10x RBC lysis buffer to 450 mL of deionized water. Store at 4 &#176;C.</li>
	<li>Collagenase IV and dispase II solution: Dissolve 30 mg of collagenase IV and 60 mg of dispase II in 30 mL of HBSS for a 1 mg/mL collagenase IV and 2 mg/mL dispase II solution. Prepare fresh before use.</li>
	<li>Fc receptors blocking solution: Add 10 &#181;L of purified anti-mouse CD16/32 antibody to 490 &#181;L of PBS 2% FBS. Prepare fresh or on the previous day. Store at 4 &#176;C until use.</li>
	<li>Fluorescent cell viability dye staining solution: Add 2 &#181;L of fluorescent cell viability dye to 1 mL of PBS. Prepare fresh before use.</li>
	<li>Biotin lineage cocktail: Mix 100 &#181;L of each of the following antibodies: biotin anti-mouse CD3&#949;, biotin anti-mouse CD4, biotin anti-mouse CD8a, biotin anti-mouse/human CD11b, biotin anti-mouse/human CD45R/B220, biotin anti-mouse Ly-6G/Ly-6C (Gr-1), and biotin anti-mouse TER-119/erythroid cells. Store at 4 &#176;C until use.</li>
	<li>HSCs primary staining cocktail: Add 10 &#181;L of biotin lineage cocktail, 10 &#181;L of immunofluorescently tagged 510 anti-mouse CD150 (SLAM), 10 &#181;L of phycoerythrin (PE) anti-mouse Flk2 (CD135), 10 &#181;L of peridinin-chlorophyll-protein (PerCP) anti-mouse Ly-6A/E (Sca-1), 10 &#181;L of PE/Cyanine7 anti-mouse CD48, and 10 &#181;L of allophycocyanin (APC)/Cyanine7 anti-mouse CD117 (c-kit) to 940 &#181;L of PBS 2% FBS. Prepare fresh or on the previous day. Store at 4 &#176;C protected from light until use.</li>
	<li>Stromal primary staining cocktail: Add 5 &#181;L of mouse leptin R biotinylated antibody, 6.7 &#181;L of PE anti-mouse endomucin antibody, 10 &#181;L of PerCP anti-mouse Ly-6A/E (Sca-1), 4 &#181;L of PE/Cyanine7 anti-mouse CD31, 10 &#181;L of APC/Cyanine7 anti-mouse CD45, and 10 &#181;L of APC/Cyanine7 anti-mouse TER-119/erythroid cells to 954 &#181;L of PBS 2% FBS. Prepare fresh or on the previous day. Store at 4 &#176;C protected from light until use.</li>
	<li>HSCs/stromal secondary staining solution: Add 1 &#181;L of APC streptavidin to 1 mL of PBS 2% FBS. Prepare fresh or on the previous day. Store at 4 &#176;C protected from light until use.</li>
	<li>ECs staining cocktail: Add 10 &#181;L of PE anti-mouse endomucin antibody, 10 &#181;L of PerCP anti-mouse Ly-6A/E (Sca-1), 10 &#181;L of PE/Cyanine7 anti-mouse CD31, 10 &#181;L of Alexa Fluor 647 anti-mouse CD54/ICAM-1, 10 &#181;L of APC/Cyanine7 anti-mouse CD45, and 10 &#181;L of APC/Cyanine7 anti-mouse TER-119/erythroid cells to 940 &#181;L of PBS 2% FBS. Prepare fresh or on the previous day. Store at 4 &#176;C protected from light until use.</li>
	<li>DAPI staining solution: Add 1 drop of DAPI reagent to 500 &#181;L of PBS. Prepare fresh before use.</li>
</ol>
2. Sample extraction
<ol>
	<li>Euthanize the animal by cervical dislocation (details of the animals used to produce the data presented in this study can be found in Supplementary Table 1). Place the animal with the belly up on a Petri dish and spray with 70% ethanol. Make a cut above the abdomen using scissors and pull away the skin all the way to the ankles.</li>
	<li>Grab one leg and, using scissors, cut at its bottom (where the joint between the leg and the hip is) to separate it from the animal. This step will also serve as a secondary confirmatory method of euthanasia. Remove the foot from the leg by cutting at the ankle. Place the leg in ice-cold PBS 2% FBS. If required, repeat the step with the other leg.</li>
	<li>Grab the hip bone and, using scissors cut behind it to separate it from the animal. Place the hip bone in ice-cold PBS 2% FBS. If required, repeat the step with the other hip bone.</li>
	<li>Place the leg(s) and hip bone(s) in a Petri dish and, using a sterile scalpel, clean the bones. Separate the tibia and femur by cutting at the knee with the scalpel. Place the clean bones in ice-cold PBS 2% FBS.</li>
</ol>
3. Sample processing for analysis of hematopoietic cell populations in total bone marrow
<ol>
	<li>Place a femur or tibia in a mortar with ice-cold PBS 2% FBS and crush the bone by gently pressing it against the wall of the mortar using the pestle. BM cells are released into the solution contained in the mortar.</li>
	<li>Pipette the resulting cell suspension up and down using a 10 mL pipette to homogenize and transfer into a 50 mL tube through a 40 &#181;m cell strainer placed on top of the tube.</li>
	<li>If the crushed bones do not look white at this point, add more PBS 2% FBS to the mortar and repeat crushing, and then pipette up and down and transfer the solution into the same 50 mL tube through the same 40 &#181;m cell strainer.</li>
	<li>Rinse the mortar with some more PBS 2% FBS and transfer the solution into the same 50 mL tube through the same 40 &#181;m cell strainer. Centrifuge the 50 mL tube at 500 x g for 5 min at 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend the cell pellet in 5 mL of RBC lysis buffer (1x) at RT by pipetting up and down several times. Following a 2 min incubation at RT, add 15 mL of PBS 2% FBS.</li>
	<li>Centrifuge the 50 mL tube at 500 x g for 5 min at 4 &#176;C. If the cell pellet still looks reddish, go back to step 3.5. If not, discard the supernatant, resuspend the pellet in 200 &#181;L of PBS, and transfer the cell suspension into a well of a 96-well V-bottom plate for staining.</li>
	<li>Centrifuge the plate at 500 x g for 3 min at 4 &#176;C. Discard the supernatant by flipping the plate onto absorbent paper and resuspend the pellet in 200 &#181;L of fluorescent dye staining solution. Incubate the plate for 15 min at RT in the dark.</li>
	<li>Centrifuge plate at 500 x g for 3 min at 4 &#176;C. Discard the supernatant by flipping the plate onto absorbent paper and resuspend the pellet in 200 &#181;L of PBS 2% FBS to wash.</li>
	<li>Centrifuge the plate at 500 x g for 3 min at 4 &#176;C, discard the supernatant by flipping the plate onto absorbent paper, and resuspend the pellet in 100 &#181;L of Fc receptors blocking solution. Incubate the plate for 10 min at 4 &#176;C protected from light.</li>
	<li>Add 100 &#181;L of PBS 2% FBS and pipette up and down a few times to wash.&#160;Centrifuge the plate at 500 x g for 3 min at 4 &#176;C, discard the supernatant by flipping the plate onto absorbent paper, and resuspend the pellet in 100 &#181;L of HSCs primary staining cocktail. Incubate the plate for 15 min at RT in the dark.</li>
	<li>Add 100 &#181;L of PBS 2% FBS and pipette up and down a few times to wash. Centrifuge the plate at 500 x g for 3 min at 4 &#176;C, discard the supernatant by flipping the plate onto absorbent paper, and resuspend the pellet in 100 &#181;L of HSCs secondary staining solution. Incubate the plate for 15 min at RT in the dark.</li>
	<li>Add 100 &#181;L of PBS 2% FBS and pipette up and down a few times to wash. Centrifuge the plate at 500 x g for 3 min at 4 &#176;C and discard the supernatant by flipping the plate onto absorbent paper.</li>
	<li>Resuspend the pellet in 250 &#181;L of PBS 2% FBS and transfer the cell suspension into a 5 mL polystyrene round-bottom tube through a 40 &#181;m cell strainer. Keep on ice protected from light until flow cytometry analysis. 
	&#8203;NOTE: If interested in determining the absolute numbers of hematopoietic cell populations, add Calibrite Beads to the polystyrene tubes before analysis in the cytometer16.</li>
</ol>
4. Sample processing for the analysis of stromal cell populations in total bone marrow
<ol>
	<li>Place a femur or tibia in a mortar with ice-cold PBS 2% FBS and crush the bone by gently pressing it against the wall of the mortar&#160;using the pestle. Cut the tip of a P1000 tip using scissors and use it to transfer all the content of the mortar into a 50 mL tube (do not pass through a cell strainer).</li>
	<li>Centrifuge the 50 mL tube at 500 x g for 5 min at 4 &#176;C, discard the supernatant, and resuspend the pellet in 3 mL of collagenase IV and dispase II solution. Incubate the tube at 37 &#176;C for 40 min.</li>
	<li>Top up the tube to 25 mL with PBS 2% FBS and vortex to mix. Transfer the content of the 50 mL tube to a new 50 mL tube through a 100 &#181;m cell strainer. Add 15 mL of PBS 2% FBS to the old 50 mL tube and transfer the solution into the same new 50 mL tube through the same cell strainer.&#160;Centrifuge at 500 x&#160;g&#160;for 5&#160;min at 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend the cell pellet in 5 mL of RBC lysis buffer (1x) at RT by pipetting up and down several times. Following a 2 min incubation at RT, add 15 mL of PBS 2% FBS.</li>
	<li>Centrifuge the 50 mL tube at 500 x g for 5 min at 4 &#176;C. If the cell pellet still looks reddish, go back to step 4.4. If not, discard the supernatant, resuspend the pellet in 200 &#181;L of PBS 2% FBS, and transfer the cell suspension into a well of a 96-well V-bottom plate for staining. Centrifuge the plate at 500 x g for 3 min at 4 &#176;C.</li>
	<li>If performing stromal staining, proceed to step 4.7. If performing EC staining, proceed to step 4.12.</li>
	<li>Discard the supernatant by flipping the plate onto absorbent paper and resuspend the pellet in 100 &#181;L of stromal primary staining cocktail. Incubate the plate for 15 min at RT in the dark.</li>
	<li>Add 100 &#181;L of PBS 2% FBS and pipette up and down a few times to wash. Centrifuge the plate at 500 x g for 3 min at 4 &#176;C, discard the supernatant by flipping the plate onto absorbent paper, and resuspend the pellet in 100 &#181;L of stromal secondary staining solution. Incubate the plate for 15 min at RT in the dark.</li>
	<li>Add 100 &#181;L of PBS 2% FBS and pipette up and down a few times to wash. Centrifuge the plate at 500 x g for 3 min at 4 &#176;C. Discard the supernatant by flipping the plate onto absorbent paper.</li>
	<li>Resuspend the pellet in 250 &#181;L of PBS 2% FBS and transfer the cell suspension into a 5 mL polystyrene round-bottom tube through a 40 &#181;m cell strainer. Keep on ice protected from light.</li>
	<li>Just before going to the cytometer, add 35 &#181;L of DAPI staining solution to the tube containing the cell suspension for flow cytometry analysis and incubate the tube at RT for 5 min in the dark. Following this incubation, keep on ice protected from light until flow cytometry analysis.</li>
	<li>Discard the supernatant by flipping the plate onto absorbent paper and resuspend the pellet in 100 &#181;L of ECs staining cocktail. Incubate the plate for 15 min at RT in the dark.</li>
	<li>Add 100 &#181;L of PBS 2% FBS and pipette up and down a few times to wash. Centrifuge the plate at 500 x g for 3 min at 4 &#176;C.</li>
	<li>Discard the supernatant by flipping the plate onto absorbent paper. Resuspend the pellet in 250 &#181;L of PBS 2% FBS and transfer the cell suspension into a 5 mL polystyrene round-bottom tube through a 40 &#181;m cell strainer. Keep on ice protected from light.</li>
	<li>Just before going to the cytometer, add 35 &#181;L of DAPI staining solution to the tube containing the cell suspension for flow cytometry analysis and incubate the tube at RT for 5 min in the dark. Following this incubation, keep on ice protected from light until flow cytometry analysis. 
	&#8203;NOTE: If interested in determining the absolute numbers of stromal and endothelial cell populations, add Calibrite Beads to the polystyrene tubes before analysis in the cytometer16.</li>
</ol>
5. Sample processing for the analysis of hematopoietic cell populations in crushed and flushed BM
<ol>
	<li>Place a femur on a Petri dish and cut the ends of the bone using a sterile scalpel.</li>
	<li>Flush the central part of the bone (diaphysis) by passing 100 &#181;L of PBS 2% FBS through the inside of the bone once from each side using a no dead volume 0.5 mL insulin syringe and collecting the solution in a microcentrifuge tube containing 1 mL of PBS 2% FBS. Subsequently, transfer the cell suspension into a 50 mL tube labeled as flushed BM containing 4 mL of PBS 2% FBS. Keep on ice.</li>
	<li>Place the ends of the bone in a mortar with ice-cold PBS 2% FBS and crush by gently pressing it against the mortar wall using the pestle. Pipette the resulting cell suspension up and down using a 10 mL pipette to homogenize and transfer into a 50 mL tube labeled as crushed BM through a 40 &#181;m cell strainer.</li>
	<li>If the crushed bone does not look white at this point, add more PBS 2% FBS to the mortar and repeat the crushing. Then pipette up and down and transfer the solution into the same 50 mL tube (crushed BM) through the same 40 &#181;m cell strainer.</li>
	<li>Rinse the mortar with some more PBS 2% FBS and transfer the solution into the same 50 mL tube (crushed BM) through the same 40 &#181;m cell strainer.</li>
	<li>Centrifuge the 50 mL tubes corresponding to the flushed and crushed samples at 500 x g for 3 min at 4 &#176;C.</li>
	<li>Follow steps 3.5 to 3.13 (section 3) with the flushed and crushed BM samples.</li>
</ol>
6. Preparation of single-color controls (SCCs) for flow cytometry analysis
<ol>
	<li>Follow steps 3.1 to 3.6 (section 3) with an extra bone from one of the animals not used for the main analysis, transferring the final cell suspension into a microcentrifuge tube containing 1 mL of PBS 2% FBS instead of a well of a 96-well V-bottom plate.</li>
	<li>Transfer 100 &#181;L of the cell suspension to 8 wells of a 96-well V-bottom plate, 100 &#181;L into a 5 mL polystyrene round-bottom tube containing 100 &#956;L of PBS 2% FBS labeled as DAPI SCC, and the remaining cell suspension into a 5 mL polystyrene round-bottom tube containing 100&#160;&#956;L of PBS 2% FBS&#160;labeled as unstained. Keep the tubes on ice protected from light.</li>
	<li>Centrifuge the plate at 500 x g for 3 min at 4 &#176;C and discard the supernatants by flipping the plate onto absorbent paper.</li>
	<li>Resuspend one pellet in 100 &#181;L of fluorescent dye staining solution and the remaining ones in 100 &#181;L of PBS 2% FBS.</li>
	<li>Add 0.5 &#181;L of the following antibodies, apart from the lineage cocktail of antibodies for which add 2 &#181;L and the PECy7 CD31 antibody for which add 0.3 &#181;L, to each of the wells with cell suspension in PBS 2% FBS (one antibody per well, same antibodies used in the main analysis panels or same fluorophore antibodies with similar fluorescence intensity and epitope abundance): biotin lineage cocktail, immunofluorescently tagged 510 anti-mouse CD150 (SLAM), PE anti-mouse Flk2 (CD135), PerCP anti-mouse Ly-6A/E (Sca-1), immunofluorescently tagged 647 anti-mouse CD54/ICAM-1, PE/Cyanine7 anti-mouse CD48, and APC/Cyanine7 anti-mouse CD117 (c-kit). Incubate the plate for 15 min at RT in the dark.</li>
	<li>Add 100 &#181;L of PBS 2% FBS to each well and pipette up and down a few times to wash. Centrifuge the plate at 500 x g for 3 min at 4 &#176;C and discard the supernatants by flipping the plate onto absorbent paper.</li>
	<li>Resuspend all the pellets apart from the one corresponding to the cells incubated with the lineage cocktail of antibodies in 180 &#181;L of PBS 2% FBS and transfer the cell suspensions into 5 mL polystyrene round-bottom tubes through 40 &#181;m cell strainers. Keep on ice protected from light until flow cytometry analysis.</li>
	<li>Resuspend the cells incubated with the lineage cocktail of antibodies in 100 &#181;L of HSCs/stromal secondary staining solution. Incubate the plate for 15 min at RT in the dark.</li>
	<li>Add 100 &#181;L of PBS 2% FBS and pipette up and down a few times to wash. Centrifuge the plate at 500 x g for 3 min at 4 &#176;C.</li>
	<li>Resuspend the pellet in 180 &#181;L of PBS 2% FBS and transfer the cell suspension into a 5 mL polystyrene round-bottom tube through a 40 &#181;m cell strainer. Keep on ice protected from light until flow cytometry analysis.</li>
	<li>Just before going to the cytometer, add 35 &#181;L of DAPI staining solution to the DAPI SCC tube and incubate the tube at RT for 5 min in the dark. Following this incubation, keep on ice protected from light until flow cytometry analysis.</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All+hematopoietic+cells+develop+from+hematopoietic+stem+cells+through+Flk2/Flt3-positive+progenitor+cells.">All hematopoietic cells develop from hematopoietic stem cells through Flk2/Flt3-positive progenitor cells.</a> Cell Stem Cell. 9 (1), 64-73 (2011).</li><li>Siegemund, S., Shepherd, J., Xiao, C., Sauer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hCD2-iCre+and+Vav-iCre+mediated+gene+recombination+patterns+in+murine+hematopoietic+cells.">hCD2-iCre and Vav-iCre mediated gene recombination patterns in murine hematopoietic cells.</a> PLoS One. 10 (4), 0124661 (2015).</li></ol>

The authors have no conflicts of interest to disclose.

While the protocol described is simple and easy to perform, special attention should be brought to specific steps. For example, when obtaining flushed BM (step 5.2), the volume or number of times indicated to pass PBS 2% FBS through the inside of the central part of the bone should not be exceeded, as this might result in significant contamination of the flushed sample by endosteal cell populations.
Alterations to the protocol can be made to facilitate its execution by the investigator. In sam...

Flow cytometry is an invaluable method to characterize and prospectively isolate immune and hematopoietic cells. It is also increasingly being used to analyze stromal and epithelial populations of different tissues. The hematopoietic stem cell (HSC) has unique properties of self-renewal and multipotency. In adult mammals, HSCs primarily reside in the bone marrow (BM), where they receive quiescence and survival signals from the surrounding microenvironment or niche1. HSCs are formally defined according to functional assays2. Nevertheless, several landmark papers have shown the usefulness of flow cytometry to identify HSCs. Through the use of limited cell surface markers, it is possible to discriminate hematopoietic populations that are highly enriched in HSCs3. Flow cytometry is, therefore, a central method in the stem cell field. It has been extensively used to evaluate the impact of putative niche cell types and niche factors on HSCs. By combining flow cytometry with imaging and functional assays, it has been shown that HSCs are critically supported by perivascular mesenchymal stem cells (MSCs) and endothelial cells (ECs). BM MSCs are a heterogenous group and have different cytokine contributions4, but it is well established that leptin receptor (LepR)+ MSCs are key niche cells1. BM ECs are also highly heterogeneous and can be part of sinusoids, arterioles, and type H/transitional vessels5. Different studies have shown the nuanced contribution of these different ECs. For example, endosteal sinusoidal ECs are spatially closer to quiescent HSCs6, while non-migratory HSCs with lower levels of reactive oxygen species are located near arteriolar ECs7. The endosteal versus central location of niches is also very important. Endosteal type H vessels are associated with perivascular stromal cells that are lost with aging, leading to the loss of HSCs8. In acute myeloid leukemia, central ECs are expanded, while endosteal vessels and endosteal HSCs are lost9.
Most studies in the field have focused on hematopoiesis itself and on the cell&#39;s extrinsic regulation of HSCs. It has been, however, increasingly recognized that there is a need to better characterize the niches that regulate other progenitors, namely multipotent progenitors (MPPs), particularly considering that they are the main drivers of hematopoiesis in steady state10. In contrast with a fixed hierarchical structure, recent studies have shown that hematopoiesis is a continuum&#160;in which HSCs differentiate into biased MPPs at an early stage11. MPPs have been named after different classification schemes12, but a recent consensus paper by the international society for experimental hematology (ISEH) proposed MPPs to be discriminated as early MPPs and according to their lymphoid (MPP-Ly), megakaryocytic and erythroid (MPP-Mk/E), and myeloid (MPP-G/M) bias13. The use of flow cytometry will be critical in further studying the importance of BM niches in the regulation of these populations. Current flow cytometry methods use variable gating strategies to differentiate HSPCs and identify stromal cells, namely ECs, using inconsistent markers. The goal of the current method is to present a simple and reproducible workflow of BM staining to identify HSPC subpopulations, heterogeneous groups of ECs, and LepR+ MSCs. We believe this technique, although comparable with previously reported methods (see, for example, reference14), provides an updated and easy-to-implement protocol for the phenotypic analysis of hematopoietic cells in the two functional marrow areas, endosteal and central BM8,15, as well as BM stromal niche cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 647 anti-mouse CD54/ICAM-1 antibody</td>
			<td>BioLegend</td>
			<td>116114</td>
			<td></td>
		</tr>
		<tr>
			<td>APC Streptavidin</td>
			<td>BioLegend</td>
			<td>405207</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>APC/Cyanine7 anti-mouse CD45 antibody</td>
			<td>BioLegend</td>
			<td>103116</td>
			<td></td>
		</tr>
		<tr>
			<td>APC/Cyanine7 anti-mouse TER-119/erythroid cells antibody</td>
			<td>BioLegend</td>
			<td>116223</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin anti-mouse CD3&#949; antibody</td>
			<td>BioLegend</td>
			<td>100304</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin anti-mouse CD4 antibody</td>
			<td>BioLegend</td>
			<td>100404</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin anti-mouse CD8a antibody</td>
			<td>BioLegend</td>
			<td>100704</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin anti-mouse Ly-6G/Ly-6C (Gr-1) antibody</td>
			<td>BioLegend</td>
			<td>108404</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin anti-mouse TER-119/erythroid cells antibody</td>
			<td>BioLegend</td>
			<td>116204</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin anti-mouse/human CD11b antibody</td>
			<td>BioLegend</td>
			<td>101204</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin anti-mouse/human CD45R/B220 antibody</td>
			<td>BioLegend</td>
			<td>103204</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 510 anti-mouse CD150 (SLAM) antibody</td>
			<td>BioLegend</td>
			<td>115929</td>
			<td></td>
		</tr>
		<tr>
			<td>Calibrite 2 Color Beads</td>
			<td>BD Biosciences</td>
			<td>349502</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Merck Life Science</td>
			<td>C1889</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Merck Life Science</td>
			<td>D4693</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, heat inactivated, E.U.-approved, South America Origin</td>
			<td>ThermoFisher Scientific</td>
			<td>10500064</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td>ThermoFisher Scientific</td>
			<td>14175095</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Leptin R Biotinylated Antibody</td>
			<td>R&#38;D systems</td>
			<td>BAF497</td>
			<td></td>
		</tr>
		<tr>
			<td>NucBlue Fixed Cell Reagent (DAPI)</td>
			<td>ThermoFisher Scientific</td>
			<td>R37606</td>
			<td>DAPI reagent</td>
		</tr>
		<tr>
			<td>PE anti-mouse endomucin antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>12-5851-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse Flk2 (CD135)</td>
			<td>ThermoFisher Scientific</td>
			<td>12-1351-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cyanine7 anti-mouse CD31 antibody</td>
			<td>BioLegend</td>
			<td>102524</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cyanine7 anti-mouse CD48 antibody</td>
			<td>BioLegend</td>
			<td>103424</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP anti-mouse Ly-6A/E (Sca-1) antibody</td>
			<td>BioLegend</td>
			<td>108122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS) tablets</td>
			<td>Merck Life Science</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified anti-mouse CD16/32 antibody</td>
			<td>BioLegend</td>
			<td>101302</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC lysis buffer 10x</td>
			<td>BioLegend</td>
			<td>420302</td>
			<td></td>
		</tr>
		<tr>
			<td>Zombie Violet Fixable Viability Dye</td>
			<td>BioLegend</td>
			<td>423114</td>
			<td>fluorescent dye</td>
		</tr>
	</tbody>
</table>

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.

Representative plots of flow cytometry analysis of HSCs and MPPs in a healthy young adult C57Bl/6 mouse are shown in Figure 1. The gating strategy follows the latest harmonizing nomenclature proposed by the ISEH13. When analyzing the impact of a perturbation, such as infection or cancer, it is important to use a control mouse as a reference for normal gates. Fluorescence-minus-one (FMO) controls can be particularly useful to delineate the boundaries of the gates, but ...

Watch this Scientific Journal Video about Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells at JoVE.com

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

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 ...

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3.5K Views.  Universidade do Porto. 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.

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

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

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 ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic homeostasis have been extensively studied by HSC researchers. Mitochondrial activity levels are reflected in their membrane potentials (&#916;&#936;m), which can be measured by cell-permeant cationic dyes such as TMRM (tetramethylrhodamine, methyl ester). The ability of efflux pumps to extrude these dyes from cells can limit their usefulness, however. The resulting measurement bias is particularly critical when assessing HSCs, as xenobiotic transporters exhibit higher levels of expression and activity in HSCs than in differentiated cells. Here, we describe a protocol utilizing Verapamil, an efflux pump inhibitor, to accurately measure &#916;&#936;m across multiple bone marrow populations. The resulting inhibition of pump activity is shown to increase TMRM intensity in hematopoietic stem and progenitor cells (HSPCs), while leaving it relatively unchanged in mature fractions. This highlights the close attention to dye-efflux activity that is required when &#916;&#936;m-dependent dyes are used, and as written and visualized, this protocol can be used to accurately compare either different populations within the bone marrow, or the same population across different experimental models.

Xenobiotic efflux pumps are highly active in hematopoietic stem and progenitor cells (HSPCs) and cause extrusion of TMRM, a mitochondrial membrane potential fluorescent dye. Here, we present a protocol to accurately measure mitochondrial membrane potential in HSPCs by TMRM in the presence of Verapamil, an efflux pump inhibitor.

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic ...