Skip to main content Skip to navigation
A series of test tubes in a laboratory.

Down Arrow Up Arrow



The extraordinary ability of stem cells to self-renew and differentiate into other types of specialized cells make them highly valuable in several therapeutic applications. This ability is critical for the preservation of tissues that constantly renew themselves, such as blood, skin and intestine. Stem cells are found in all developmental stages from the embryo (embryonic stem cells) to adulthood (adult stem cells). Several types of stem cells emerge during the developmental process and actively participate in tissue homeostasis.



Stem cells and stem cell niches

Different types of stem cells, including totipotent, pluripotent, oligopotent and unipotent stem cells, have been described. Pluripotent stem cells, e.g., embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are extensively used in stem cell research. ESCs can differentiate in culture into the three germ layers of embryonic development. Using key transcription factors, mature differentiated—or somatic—cells can be induced into pluripotent stem cells capable of differentiation into many types of cells, including hematopoietic cells (HSCs).2

Diagram detailing embryonic stem cell differentiation process.

Embryonic stem cell differentiation.

Mesenchymal stem cells (MSCs), also defined as multipotent mesenchymal stromal cells, are the predecessors of mesenchymal tissues, such as bone and cartilage. They regulate several immune functions such as the antigen presentation activity of dendritic cells, cytotoxicity of natural killer (NK) cells, and antibody production by B-cells. Due to their strong immunosuppressive function, MSCs are potential candidates for cell-based approaches for the treatment of inflammatory and autoimmune diseases. Several studies suggest the ability of MSCs to also differentiate into cell types of an unrelated tissue.3 MSCs and their derivatives encompass several types of cells that constitute the HSC niche, such as pericytes, bone-marrow stromal cells and osteo-lineage cells. MSCs can be isolated from several adult tissues (bone marrow, peripheral blood, adipose tissue) or fetal tissues and have been utilized in cell therapy research and regenerative medicine.4

Diagram detailing pluripotent stem cell differentiation process.

 Differentiation of induced pluripotent stem cells.

Stem cell niches

A stem cell niche is a nurturing environment where stem cells are regrouped, can self-renew and remain in an undifferentiated state. With appropriate cellular signaling, stem cells can be mobilized from the niche to target regions where they are required. Many stem cell niches have been identified throughout the human body, including subventricular and subgranular zones in the brain (SVZ and SGZ), hair follicles, intestinal crypts and bone marrow.5, 6, 7

Diagram showing the process for self-renewing HSC propogation.

Multicolor flow cytometry for stem cell research

Flow cytometry is a powerful tool for monitoring the heterogeneity of subsets of cells based on established markers. You can use fluorochrome-conjugated antibodies specific for either cell surface or intracellular biomarkers to verify that stem cells have maintained pluripotency. Since stem cells differentiate into the three primary germ layers and into differentiated tissue, antibodies can monitor their changing expression patterns. BD Lyoplate™ Cell Surface Marker Screening Panels provide a powerful method for discovering surface marker signatures that can be used to explore these cells in depth.


BD Biosciences offers a diverse set of tools, including high-quality antibodies, kits and cocktails, buffers, protocols, and instrumentation to support stem cell research, for characterizing, analyzing and sorting heterogeneous stem cell populations.


  1. Zakrzewski W, Dobrzyński M, Szymonowicz M, Rybak Z. Stem cells: past, present, and future. Stem Cell Res Ther. 2019;10(1):68. doi:10.1186/s13287-019-1165-5

  2. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663-676. doi: 10.1016/j.cell.2006.07.024

  3. Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in health and disease. Nature Rev Immunol. 2008; 8(9):726-736. doi: 10.1038/nri2395

  4. Vizoso FJ, Eiro N, Cid S, Schneider J, Perez-Fernandez R. Mesenchymal stem cell secretome: Toward cell-free therapeutic strategies in regenerative medicine. Int J Mol Sci. 2017;18(9):1852. doi: 10.3390/ijms18091852

  5. Boulais PE, Frenette PS. Making sense of hematopoietic stem cell niches. Blood. 2015;125(17):2621-2629. doi:10.1182/blood-2014-09-570192

  6. Ferraro F, Celso CL, Scadden D. Adult stem cels and their niches. Adv Exp Med Biol. 2010;695:155-168. doi:10.1007/978-1-4419-7037-4_11

  7. Cable J, Fuchs E, Weissman I, et al. Adult stem cells and regenerative medicine-a symposium report. Ann N Y Acad Sci. 2020;1462(1):27-36. doi:10.1111/nyas.14243


Surface Staining
Down Arrow Up Arrow

Surface staining

BD Biosciences offers effective solutions to prepare stem cells without altering the expression of surface protein epitopes. 


Sample preparation

Stem cells tend to be adherent and can grow as three-dimensional structures. To prepare a single-cell suspension for flow cytometric analysis, enzymatic digestion (with BD® Accutase™ Cell Detachment Solution or trypsin) or mechanical scraping can be used. Since enzymatic methods might cleave or modify some protein epitopes during the digestion process, preventing antibody labeling, they must be evaluated for each surface marker being measured. BD® Accutase™ Solution tends to be more broadly applicable than trypsin and scraping.


Monoclonal antibodies

Once cells are harvested and the dissociation buffer is removed, the cells are ready to be stained with antibodies. BD Biosciences offers an extensive reagent selection of antibodies against hundreds of stem cell markers conjugated to a variety of fluorochromes for flexibility in experimental design. For analysis of rare events and low-density antigens, BD Horizon Brilliant Violet™ Dyes can increase brightness and resolution. For ease of use, BD Stemflow™ Kits and Cocktails contain standard antibody panels for analysis or sorting of your stem cells.


Representative markers of selected stem cells and derivatives

Human Markers Mouse Markers BD Stemflow™️ or Other Kit Cat. No.
Alkaline Phosphate, SSEA-4, SSEA-3,
TRA-1-81, TRA-1-60

Human iPSC Sorting and Analysis Kit 562626
Human Pluripotent Stem Cell Sorting and Analysis Kit 560461
Human and Mouse Pluripotent Stem Cell Analysis Kit 560477
Hematopoietic Stem Cells (HSCs)
CD34, CD49f, CD90

CD38, CD45RA, Lineage*
CD150, c-Kit, Sca1

CD34, CD41, CD48, Lineage
BD Pharmingen™️ Human Lineage Cocktail 4 562722
Mouse Hematopoietic Stem Cell Isolation Kit 560492
Mesenchymal Stem Cells (MSCs)
CD44, CD73, CD90,
CD105, CD146, CD271

CD11b, CD19, CD31,
CD34, CD45, CD144,
CD29, CD44, CD90,
CD105, CD106, Sca-1

CD11b, CD31, CD45,
Human MSC Analysis Kit 562245
Human Mesenchymal Stem Cell Lineage Antibody Cocktail 562530
Neural Stem Cells (NSCs)
CD15mid, CD24, CD184

CD44, CD271
CD15low, CD24

CD44, CD184
Human Neural Cell Sorting Kit 562271
CD15, CD24, CD34,
CD44, CD45, CD49f,
CD166, CD326, CD338,
Her-2/Neu, Lgr5

* Human lineage (lin) markers: CD2, CD3, CD4, CD7, CD8, CD10, CD11b, CD14, CD19, CD20, CD56, CD235a.

Hematopoietic stem cell phenotyping

Cells of the hematopoietic system are well characterized with respect to surface marker expression, which is often used to isolate and characterize subsets of cells during hematopoiesis. Hematopoietic stem cells (HSCs), the source of these hematopoietic cells, are currently a focus area in stem cell biology because they can be used to replenish normal bone marrow function.


Historically, among a pool of cells, HSCs were identified as lineage-negative cells that expressed CD90 and CD34.1 Additional markers to enrich pools of long-term HSCs (LT-HSCs) capable of self-renewal include CD38,2 CD45RA,3 and CD49f.4 Reportedly, about 10% of cells with a Lin–CD34+CD38–CD90+CD45RA–CD49f+ phenotype are able to provide long-term repopulating capacity in mouse models.4


Data sets showing LT HSC gating strategy and various test results.


  1. Baum CM, Weissman IL, Tsukamoto AS, Buckle AM, Peault B. Isolation of a candidate human hematopoietic stem cell population. Proc Natl Acad Sci U S A. 1992;89(7):2804-2808. doi: 10.1073/pnas.89.7.2804

  2. Bhatia M, Wang JC, Kapp U, Bonnet D, Dick JE. Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci U S A. 1997;94(10):5320- 5325. doi: 10.1073/pnas.94.10.5320

  3. Majeti R, Park CY, Weissman IL. Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood. Cell Stem Cell. 2007;1(6):635-645. doi: 10.1016/j.stem.2007.10.001

  4. Notta F, Doulatov S, Laurenti E, Poeppl A, Jurisica I, Dick JE. Isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment. Science. 2011;333(6039):218-221. doi: 10.1126/science.1201219
Intracellular Staining
Down Arrow Up Arrow

Intracellular staining

Intracellular staining allows you to extend the speed and statistical relevance of flow cytometry to the investigation of functional proteins inside the cell. It can be used in combination with surface staining to identify critical time points, markers and proportions of cells moving along particular differentiation pathways. Intracellular staining protocols require the cells to be fixed and permeabilized so that antibodies can access the cytoplasm and nucleus. Since fixation effectively kills the cells, intracellular staining is not compatible with live-cell sorting.


Sample preparation

For intracellular staining, as with surface staining, a single-cell suspension must be prepared using enzymatic or mechanical methods. We recommend the BD® Accutase™ Solution since it helps to prevent cell clumping and can preserve surface proteins for simultaneous analysis. After optional surface staining, cells must be fixed and permeabilized to enable antibodies to enter. The cells are then stained with fluorescent-labeled antibodies to intracellular antigens and analyzed on a flow cytometer.


To optimize permeabilization and staining conditions, BD has developed several kits for the detection of key stem cell transcription factors. The kits contain optimized antibodies and buffer systems to characterize stem cells as well as their differentiation into various lineages.

Cell Type Intracellular Markers BD Stemflow™ Kit Cat. No.
Embryonic stem cells (ESCs)

Induced pluripotent stem cells (IPSCs)
Nanog, Oct 3/4, Sox2 Human Pluripotent Stem Cell Transcription Factor Analysis Kit 560589
Mouse Pluripotent Stem Cell Transcription Factor Analysis Kit 560585
Neural stem cells (NSCs) Nestin, Pax6, Sox1, Sox2 Human Neural Lineage Analysis Kit 561526
Astrocytes GFAP Human Neural Lineage Analysis Kit 561526
Neurons Doublecortin Human Neural Lineage Analysis Kit 561526
Early pancreatic endoderm FoxA2, Pax6, Pdx1, Sox17 Human Definitive and Pancreatic Endoderm Analysis Kit 562496
Late pancreatic endoderm NeuroD1, Nkx6.1    
Cardiac cTNI, GATA4, Islet-1, Myosin Heavy Chain    
Hepatic AFP, GATA4    

Stem cell differentiation

The ability of human pluripotent stem cells to differentiate into various cell lineages is a central topic in developmental biology and has applications for regenerative medicine and cellular therapy. As pluripotent cells differentiate into different lineages, the expression of transcription factors and other proteins can change. Multiparametric flow cytometry is an excellent method for determining the relative numbers of cells expressing markers of interest and can be used to optimize, quantitate and compare differentiation protocols and differentiation potential.


For example, in mammalian embryonic development, the definitive endoderm generates the liver, pancreas and intestine.1 During lineage specification into definitive endoderm, the levels of transcription factors Sox17 and FoxA2 increase, while pluripotency markers such as Nanog decrease.2

Data set showing day zero and day 3 results for FoxA2 and Nanog tests.

The differentiation of neural stem cells to neural lineages can also be monitored using multicolor flow analysis. As neural stem cells (NSCs) differentiate into neurons, they gradually express less Nestin and more of the early neuronal marker doublecortin (DCX). A subpopulation of cells that continues to express Nestin further delineates into a glial cell population that expresses CD44.


Rapid marker screening

As the leader in flow cytometry technologies, BD Biosciences offers robust tools to efficiently support your screening efforts.


A major challenge facing stem cell biology is the heterogeneous nature of cultures and differentiations. To sort viable cells to purify for use in later experiments, one must know the cell surface signature for the cell of interest. Since different types of related cells may share markers, researchers must find a unique multimarker signature for each, while other markers may be useful in distinguishing subpopulations of a particular type of cell.

Data sets showing CD44 and DCX results after 0, 14, and 27 day periods.

BD Lyoplate™ Cell Surface Marker Screening Panels provide a comprehensive and efficient solution for profiling stem cells and their derivatives for hundreds of human or mouse cell surface markers by flow cytometry or cellular imaging. Deciphering the cell surface proteome enables researchers to define strategies for the analysis and isolation of targeted cells from heterogeneous populations for functional studies, drug screening, in vivo animal studies and cell therapy research.


The hundreds of monoclonal antibodies in each panel constitute one of the most cost-effective screening tools available for cellular analysis. To simplify the transition to more targeted, larger-scale experiments, all antibodies included in the screening panels are available in the BD Biosciences catalog.


Both the human (Cat. No. 560747) and mouse (Cat. No. 562208) panels contain three plates. Each well contains lyophilized, purified antibody to one cell surface marker or isotype control. Following reconstitution, the cellular samples are stained with purified antibodies, and detection reagents included with the panel are added. Finally, samples are analyzed by flow cytometry or imaging.


To provide flexibility while simplifying workflow, open wells allow the panel to be expanded to include additional markers. Powerful BD Biosciences analysis tools facilitate data mining and heatmap generation.


Considerations for flow cytometry vs image screening with BD Lyoplate™ Panels

Property of sample Flow cytometry Imaging
Suspension cells X  
Rare cell populations X  
Subpopulation analysis

Co-staining with multiple markers 

Reporter lines X X
Specific morphology changes   X
Limited number of cells   X


  1. Murry CE, Keller G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development.  2008;132(4):661-680. doi: 10.1016/j.cell.2008.02.008

  2. D’Amour KA, Agulnick AD, Eliazer S, Kelly OG, Kroon E, Baetge EE. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol. 2005;23(12):1534-1541. doi: 10.1038/nbt1163
Down Arrow Up Arrow

Screening of neural populations

Neural cell populations derived from pluripotent stem cells are important for studying human disease and development. Pluripotent stem cells can be differentiated into self-renewing NSCs, which can be further differentiated into heterogeneous populations of neurons and glia.1 A key to further research is to identify surface marker signatures for each of these cell types.


In the example, the BD Lyoplate™ Human Cell Surface Marker Screening Panel (Cat. No. 560747) was used to identify cell surface phenotypes for NSCs and neurons. In panel A, heterogeneous neural induction cultures were screened by flow cytometry, and potential NSC markers were identified on a heatmap. A resulting NSC cell surface phenotype of CD184+CD44–CD271–CD24+CD15mid was verified using intracellular NSC markers. The surface phenotype was used to sort a near-pure subpopulation of NSCs, the ability of which to differentiate both in vivo and in vitro was later confirmed.

Diagram showing heterogeneous neural induction of cells and staining.

The purified NSCs were differentiated into neuronal and glial cell populations, which were screened by imaging using the same panel to identify surface markers for isolating neurons. An imaging screen was chosen due to the unique morphology of neurons and the ability to co-stain with a neuronal-specific marker. A potential neuronal surface phenotype of CD44CD184CD24+CD15low was verified by flow cytometry and used to purify neurons. In addition to neural cells, the BD Lyoplate™ Human Cell Surface Marker Screening Panel has also been used to identify cell surface markers of cardiomyocytes derived from pluripotent stem cells.2 This powerful methodology was used to develop a human stem cell model of Alzheimer’s disease.3





Cell stainings showing heterogeneous neuronal differentiation and testes resulting in near-pure neurons.


  1. Yuan SH, Martin J, Elia J, et al. Cell surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PLoS One. 2011;6(3):e17540. doi: 10.1371/journal.pone.0017540

  2. Uosaki H, Fukushima H, Takeuchi A, et al. Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression. PLoS One. 2011;6(8):e23657. doi: 10.1371/journal.pone.0023657

  3. Israel MA, Yuan SH, Bardy C, et al. Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells. Nature. 2012;482(7384):216-220. doi: 10.1038/nature10821
Down Arrow Up Arrow




Expand All Collapse All

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

Accutase is a trademark of Innovative Cell Technologies Corporation.

BD flow cytometers are Class 1 Laser Products.