Each type of stem cell or derivative expresses characteristic surface and intracellular proteins that can be used for identification. Because intracellular analysis requires permeabilization, surface markers are essential when researchers want to isolate live cell populations for further analysis. Fluorescence-activated cell sorting, or FACS, can be used to sort cells of interest in bulk or in single-cell depositions for downstream applications. To analyze cells for surface marker expression, a single-cell suspension must be stained with fluorescent-labeled antibodies and analyzed or sorted on a flow cytometer.
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 tends to be more broadly applicable than trypsin and yields a more consistent single-cell suspension than does 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, the BD Horizon™ Brilliant Violet™ family of reagents can increase brightness and resolution. For ease of use, BD Stemflow™ kits and cocktails contain standard antibody panels for analysis or sorting of different stem cell types.
Representative surface markers of selected stem cells and derivatives
Human Markers |
Mouse Markers |
BD Stemflow™ or Other Kit |
Cat. No. |
Pluripotent Stem Cells (ESCs and iPSCs) |
Positive: Alkaline Phosphatase, SSEA-4, SSEA-3, TRA-1-81, TRA-1-60
Negative: SSEA-1
|
Positive: SSEA-1 |
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) |
Positive: CD34, CD49f, CD90
Negative: CD38, CD45RA, Lineage*
|
Positive: CD150, c-Kit, Sca1
Negative: CD34, CD41, CD48, Lineage
|
BD Pharmingen™ Human Lineage Cocktail 4 |
562722 |
Mouse Hematopoietic Stem Cell Isolation Kit |
560492 |
Mesenchymal Stem Cells (MSCs) |
Positive: CD44, CD73, CD90, CD105, CD146, CD271
Negative: CD11b, CD19, CD31, CD34, CD45, CD144, HLA-DR
|
Positive: CD29, CD44, CD90, CD105, CD106, Sca-1
Negative: CD11b, CD31, CD45, Ter-119
|
Human MSC Analysis Kit |
562245 |
Human Mesenchymal Stem Cell Lineage Antibody Cocktail |
562530 |
Neural Stem Cells (NSCs) |
Positive: CD15 mid, CD24, CD184
Negative: CD44, CD271
|
– |
Human Neural Cell Sorting Kit |
562271 |
Neurons |
Positive: CD15 low, CD24
Negative: CD44, CD184
|
– |
Human Neural Cell Sorting Kit |
562271 |
Cancer |
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 Recently, researchers have used additional markers to enrich pools of long-term HSCs (LT-HSCs) capable of self renewal. These markers include CD38, 2 CD45RA, 3 and most recently 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