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><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&amp;epsilon; 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&#039; 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&amp;amp;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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3.9K Views.  Universidade do Porto. Het beschreven protocol maakt de fenotypische analyse van beenmerg-hematopoëtische stam- en voorlopercellen en beenmergstromale cellen mogelijk. Het kan worden gebruikt om verstoringen van deze populaties in verschillende omgevingen te bestuderen. In vergelijking met andere eerder beschreven methoden, maakt deze techniek de fenotypische analyse van hematopoëtische cellen mogelijk, met name in de twee functionele merggebieden, endosteal en centraal beenmerg, evenals beenmergstromale cellen.

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

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Developmental Biology

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to reestablish blood cell production for the lifespan of the recipient. This protocol explains how to set up a functional assay to compare the HSC capacities of two different populations of cells, such as bone marrow from mice of two different genotypes, and how to analyze the recipient mice by flow cytometry. The protocol uses HSC equivalents rather than cell sorting for standardization and discusses the advantages and disadvantages of both approaches. We further discuss different variations to the basic protocol, including serial transplants, limiting dilution assays, homing assays and non-competitive transplants, including the advantages and preferred uses of these varied approaches. These assays are central for the study of HSC function and could be used not only for the investigation of fundamental HSC intrinsic aspects of biology but also for the development of preclinical assays for bone marrow transplant and HSC expansion in culture.

This protocol provides step-by-step guidelines for setting up competitive mouse bone marrow transplant experiments to study hematopoietic stem/progenitor cell function without prior purification of stem cells by cell sorting.

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to ...

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a differentiation state to sustain demands of the blood formation. However, it has been difficult to analyze the metabolic status (mitochondrial respiration and glycolysis) of HSPCs due to their limited numbers and lack of optimized protocols for non-adherent, fragile HSPCs. Here, we provide a set of clear, step-by-step instructions to measure metabolic respiration (oxygen consumption rate; OCR) and glycolysis (extracellular acidification rate; ECAR) of murine bone marrow-LineagenegSca1+c-Kit+ (LSK) HSPCs. This protocol provides a higher amount of LSK HSPCs from murine bone marrow, improves the viability of HSPCs during incubation, facilitates extracellular flux analyses of non-adherent HSPCs, and provides optimized injection protocols (concentration and time) for drugs targeting oxidative phosphorylation and glycolytic pathways. This method enables the prediction of the metabolic status and the health of HSPCs during blood development and diseases.

Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs.

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a ...

Isolation and Characterization of Bone Marrow Microenvironment in Myeloid Neoplasms

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

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

Author Spotlight: Analyzing Bone Marrow Microenvironment in Murine Hematological Malignancies 

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

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

Cancer Research

Identification and Isolation of 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 ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow cytometry (MFC) for myeloid disease diagnosis in France. The protocol presented here was agreed-upon and applied between September 2013 and November 2015 in six French diagnostic laboratories (University Hospitals of Saint-Etienne, Grenoble, Clermont-Ferrand, Nice, and Lille and Institut Paoli-Calmettes in Marseille) and allowed the standardization of bone marrow sample preparation and data acquisition. Three maturation databases were developed for neutrophil, monocytic, and erythroid lineages with bone marrow from "healthy" donor individuals (individuals without any evidence of a hematopoietic disease). A robust method of analysis for each myeloid lineage should be applicable for routine diagnostic use. New cases can be analyzed in the same manner and compared against the usual databases. Thus, quantitative and qualitative phenotypic abnormalities can be identified and those above 2SD compared with data of normal bone marrow samples should be considered indicative of pathology. The major limitation is the higher variability between the data achieved using the monoclonal antibodies obtained with the methods based on hybridoma technologies and currently used in clinical diagnosis. Setting criteria for technical validation of the data acquired may help improve the utility of MFC for MDS diagnostics. The establishment of these criteria requires analysis against a database. The reduction of investigator subjectivity in data analysis is an important advantage of this method.

The MDS diagnosis is difficult in the absence of morphological criteria or non-informative cytogenetics. MFC could help refine the MDS diagnostic process. To become useful for clinical practice, the MFC analysis must be based on parameters with sufficient specificity and sensitivity, and data should be reproducible between different operators.

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow ...

Medicine

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 ...

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able to generate large datasets, necessitate the destruction of the cell of interest, making it impossible to assess its functional fate. Heterogeneous biological differentiating systems like hematopoiesis are therefore difficult to describe. Building on cell division tracking dyes, we further developed a protocol to simultaneously determine kinship, division number, and differentiation status for many single hematopoietic progenitors. This protocol allows the assessment of the ex vivo differentiation potential of murine and human hematopoietic progenitors, isolated from various biological sources. Moreover, as it is based on flow cytometry and a limited number of reagents, it can quickly generate a large amount of data, at the single-cell level, in a relatively inexpensive manner. We also provide the analytical pipeline for single-cell analysis, combined with a robust statistical framework. As this protocol allows the linking of cell division and differentiation at the single-cell level, it can be used to quantitatively assess symmetric and asymmetric fate commitment, the balance between self-renewal and differentiation, and the number of divisions for a given commitment fate. Altogether, this protocol can be used in experimental designs aiming to unravel the biological differences between hematopoietic progenitors, from a single-cell perspective.

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Presented here is a flow cytometry-based technique that allows for simultaneously measuring the number of cell divisions, surface cell phenotype, and cellular kinship. Those properties can be tested statistically using a permutation-based framework.

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able ...

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

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