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 a...

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+lymphocyte+proliferation,+survival+and+differentiation+using+CFSE+time-series+data.">Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data.</a> Nature Protocols. 2 (9), 2057-2067 (2007).</li><li>Akinduro, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+dynamics+of+acute+myeloid+leukaemia+and+haematopoietic+progenitors+competing+for+bone+marrow+space.">Proliferation dynamics of acute myeloid leukaemia and haematopoietic progenitors competing for bone marrow space.</a> Nature Communications. 9 (1), 519 (2018).</li><li>Beerman, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionally+distinct+hematopoietic+stem+cells+modulate+hematopoietic+lineage+potential+during+aging+by+a+mechanism+of+clonal+expansion.">Functionally distinct hematopoietic stem cells modulate hematopoietic lineage potential during aging by a mechanism of clonal expansion.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (12), 5465-5470 (2010).</li><li>Gomariz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+spatial+analysis+of+haematopoiesis-regulating+stromal+cells+in+the+bone+marrow+microenvironment+by+3D+microscopy.">Quantitative spatial analysis of haematopoiesis-regulating stromal cells in the bone marrow microenvironment by 3D microscopy.</a> Nature Communications. 9 (1), 2532 (2018).</li><li>Xu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+factor+is+selectively+secreted+by+arterial+endothelial+cells+in+bone+marrow.">Stem cell factor is selectively secreted by arterial endothelial cells in bone marrow.</a> Nature Communications. 9 (1), 2449 (2018).</li><li>Amend, S. 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(110), e53936 (2016).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ephrin-B2+controls+VEGF-induced+angiogenesis+and+lymphangiogenesis.">Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis.</a> Nature. 465 (7297), 483-486 (2010).</li><li>DeFalco, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-assisted+mapping+of+neural+inputs+to+a+feeding+center+in+the+hypothalamus.">Virus-assisted mapping of neural inputs to a feeding center in the hypothalamus.</a> Science. 291 (5513), 2608-2613 (2001).</li><li>Boyer, S. W., Schroeder, A. V., Smith-Berdan, S., Forsberg, E. 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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 ...

The described protocol allows for the phenotypic analysis of bone marrow hematopoietic stem and progenitor cells and bone marrow stromal cells. It can be used to study perturbations to these populations in different settings. Compared with other previously described methods, this technique allows for the phenotypic analysis of hematopoietic cells, specifically in the two functional marrow areas, endosteal and central bone marrow, as well as bone marrow stromal cells.To begin, place the euthanized 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. Grab one of the legs and cut at its bottom to separate it from the animal using scissors.Then remove the foot from the leg by cutting at the ankle and place the leg in ice cold PBS 2%FBS. Next, grab the hip bone using tweezers and cut behind it to separate it from the animal using scissors. Place the hip bone in ice cold PBS 2%FBS.After placing the leg and hip bone in a Petri dish, clean the bones using a sterile scalpel. Then separate the tibia and femur by cutting at the knee with the scalpel and place the clean bones in ice cold PBS 2%FBS. Place the 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.Next, pipette the resulting cell suspension up and down using a 10 milliliter pipette to homogenize and transfer it into a 50 milliliter tube through a 40 micrometer cell strainer placed on top of the tube. Rinse the mortar with some more PBS 2%FBS and transfer the solution into the same 50 milliliter tube through the same 40 micrometer cell strainer. Centrifuge the 50 milliliter tube at 500 G for five minutes at four degrees Celsius.After discarding the supernatant, resuspend the cell pellet in five milliliters of RBC lysis buffer at room temperature by pipetting up and down several times. Following a two-minute incubation at room temperature, add 15 milliliters of PBS 2%FBS and centrifuge the 50 milliliter tube. If the cell pellet does not look reddish, discard the supernatant, resuspend the pellet in 200 microliters of PBS 2%FBS and transfer the cell suspension into a well of a 96-well V bottom plate for staining.After crushing the bone as demonstrated previously, cut the tip of a P1000 pipette using scissors and use it to transfer all the content of the mortar into a 50 milliliter tube. Then centrifuge the 50 milliliter tube at 500 G for five minutes at four degrees Celsius. After discarding the supernatant, resuspend the pellet in three milliliters of collagenase IV and this dispase-II solution.Incubate the tube at 37 degrees Celsius for 40 minutes. Top up the tube to 25 milliliters with PBS 2%FBS and vortex to mix. Next, transfer the content to a new 50 milliliter tube through a 100 micrometer cell strainer.Then add 15 milliliters of PBS 2%FBS to the old 50 milliliter tube and transfer the solution into the new 50 milliliter tube through the 100 micrometer cell strainer. After centrifuging and discarding the supernatant, resuspend the cell pellet in five milliliters of RBC lysis buffer by pipetting up and down several times with a pipette. Following the two-minute incubation at room temperature, add 15 milliliters of PBS 2%FBS.Again, centrifuge the tube and if the cell pellet does not look reddish after discarding the supernatant, resuspend the pellet in 200 microliters of PBS 2%FBS and transfer the cell suspension into a well of a 96-well V bottom plate for staining. Then centrifuge the plate at 500 G for three minutes at four degrees Celsius. Place the femur on a Petri dish and cut the ends of the bone using a sterile scalpel.Then flush the central part of the bone by passing 100 microliters of PBS 2%FBS through the inside of the bone once from each side using a no dead volume 0.5 milliliter insulin syringe and collecting the solution in a microcentrifuge tube containing one milliliter of PBS 2%FBS. Subsequently transfer the cell suspension into a 50 milliliter tube labeled as flushed bone marrow containing four milliliters of PBS 2%FBS. Crush the ends of the bone in a mortar with ice cold PBS 2%FBS by gently pressing them against the wall of the mortar using the pestle.Once the cell suspension is homogenized, transfer it into a 50 milliliter tube labeled as crushed bone marrow through a 40 micrometer cell strainer. At last, rinse the mortar with some more PBS 2%FBS and transfer the solution into the same tube. Flow cytometry analysis of hematopoietic stem cells and multipotent progenitors was performed in total bone marrow in a healthy young adult C57BL6J mouse.In the analysis of stromal cells, endothelial cells, and mesenchymal stem cells in total bone marrow, mesenchymal stem cells can be readily identified based on leptin receptor expression and endothelial cells can be identified based on the expression of CD31 and SCA-1. Flow cytometry analysis of endothelial cells in total bone marrow showing the discrimination between arteriolar and sinusoidal endothelial cells based on ICAM1. The quantification of arterial and sinusoidal bone marrow endothelial cells in central and endosteal bone marrow tissue shows that arterial bone marrow endothelial cells are more abundant in endosteal regions, while sinusoids are more frequent in central bone marrow areas.When obtaining flushed bone marrow, the volume or number of times indicated to pass PBS 2%FBS through the inside of central part of the bone should not be exceeded. Combining the described method with functional assays of the cell populations phenotypically analyzed would provide further valuable information about the potential perturbations to these populations occurring in the studied conditions. The described protocol allows the phenotypic analysis of hematopoietic cells differentially in endosteal and central bone marrow, enabling researchers to investigate perturbations to hematopiesis potentially occurring specifically in these bone marrow locations.

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3.2K visualizações.  Universidade do Porto. O protocolo descrito permite a análise fenotípica de células-tronco hematopoéticas e progenitoras da medula óssea e células estromais da medula óssea. Ele pode ser usado para estudar perturbações nessas populações em diferentes cenários. Comparada com outros métodos previamente descritos, esta técnica permite a análise fenotípica de células hematopoéticas, especificamente nas duas áreas funcionais da medula, endosteal e medula óssea central, bem como células estromais da ...