Mouse Mesenchymal Stem Cell Multi-Color Flow Kit
R&D Systems, part of Bio-Techne | Catalog # FMC003
Key Product Details
Summary for Mouse Mesenchymal Stem Cell Multi-Color Flow Kit
Kit Summary
Why is it important to verify MSC identity?
Mesenchymal stem/stromal cells (MSCs) can be isolated by a variety of techniques, grown and expanded using numerous reagents, and differentiated with a range of media, proteins, and small molecules. Variations in techniques as well as differences in the MSC starting population may account for experimental variability and some of the contradictory data in the literature.
By clearly defining the starting cell populations, researchers can decrease experimental variability and ultimately decrease apparent discrepancies in the literature that stem from the use of phenotypically different cell populations. R&D System offers the Mouse Mesenchymal Stromal Cell 4-Color Flow Cytometry Kit which is used to define mouse MSCs based on established markers. The Mouse MSC 4-Color Flow Cytometry Kit includes four fluorochrome-conjugated antibodies for the detection of MSC markers by flow cytometry.
The Mouse Mesenchymal Stromal Cell 4-Color Flow Cytometry Kit:
- Efficiently verifies MSC identity using flow cytometry.
- Defines the starting population which can reduce experimental variability.
- Includes conjugated-antibodies to reduce hands-on time.
- Includes multiple MSC markers to increase confidence in MSC identity.
Mesenchymal Stromal Cells or Mesenchymal Stem Cells?
The term ‘mesenchymal stromal cells’ is commonly used to describe a heterogeneous population of cultured cells that are adherent to plastic, have a distinct morphology, and express a specific set of marker proteins. Within this heterogeneous population are cells referred to as ‘mesenchymal stem cells.’ See Details
Mesenchymal stem cells are multipotent, self-renewing cells that have the ability to differentiate into adipocytes, chondrocytes, and osteoblasts when cultured in vitro.
2006 Proposed Change to MSC Nomenclature
Although mesenchymal stromal cells were once referred to as ‘mesenchymal stem cells,’ a change to ‘mesenchymal stromal cells’ was proposed by the International Society for Cellular Therapy in 2006.1
The change in nomenclature originates from two important factors:
- Methods used to isolate mesenchymal stem cells yield a heterogeneous population of cells with only a fraction of these cells demonstrating multipotency.
- The absence of direct evidence that mesenchymal stem cells can self-renew and differentiate in vivo.
Use of Mesenchymal Stem and Stromal Cell Terminology
Data supporting MSC self-renewal and multipotency have been obtained using in vitro conditions, which does not adequately reflect the in vivo environment. The lack of in vivo data has led some researchers to question the validity of the term ‘mesenchymal stem cell’ providing further support for the use of ‘mesenchymal stromal cells’ to describe MSCs.2 While ‘mesenchymal stromal cells’ may be the more scientifically accurate term for MSCs, the two terms are often used interchangeably in the literature. R&D Systems recognizes the use of both mesenchymal stem cells and mesenchymal stromal cells and uses ‘MSC’ to indicate mesenchymal stem/stromal cells to account for both designations.
Definitions of Mesenchymal Stromal Cells and Mesenchymal Stem Cells
- Mesenchymal Stromal Cells – A heterogeneous population of cultured cells with similar characteristics such as the ability to adhere to plastic and the expression of specific marker proteins.
- Mesenchymal Stem Cells – A subpopulation of mesenchymal stromal cells that have the capacity to self-renew and differentiate into mesodermal lineages when cultured in vitro. The capacity to self-renew and differentiate in vivo has yet to be clearly demonstrated for mesenchymal stem cells.
References
- Dominici, M. et al. (2006) Cytotherapy 8:315.
- Keating, A. (2012) Cell Stem Cell 10:709.
Kit Components
The Mouse Mesenchymal Stromal Cell 4-Color Flow Cytometry Kit contains four conjugated monoclonal antibodies and corresponding isotype controls that can be used for single-step staining of mouse mesenchymal stem/stromal cells. This kit also contains Staining Buffer (100 mL). This kit includes sufficient reagents for 25 assays. See Details
- Flow Cytometry Staining Buffer (100 mL)
Positive Markers
- Rat IgG2A Anti-Mouse Endoglin/CD105-Fluorescein (Clone 209701)
- Rat IgG2A Anti-Mouse Integrin beta 1/CD29-PE (Clone 265917)
- Rat IgG2A Anti-Mouse Sca-1-APC (Clone 177228)
Negative Markers
- Rat IgG2B Anti-Mouse CD45-PerCP (Clone 30-F11)
Stability and Storage
Store at 2 °C to 8 °C in the dark. Use within 6 months of receipt.
Precautions
The Staining Buffer contains 0.1% sodium azide. Sodium azide may react with lead and copper plumbing to form explosive metallic azides. Flush with large volumes of water during disposal.
The term 'mesenchymal stem cells' (MSCs) is most commonly used to describe multipotent self-renewing cells that can be differentiated in vitro to generate adipocytes, chondrocytes, and osteoblasts. However, because these biological properties and hierarchical relationships remain to be clearly demonstrated in vivo, the term 'multipotent mesenchymal stromal cells' is often used to distinguish cultured cells from their in vivo precursors. Originally discovered in mouse bone marrow, multipotent mesenchymal stromal cells cultured from a variety of species and tissue types, have been shown to differentiate into progeny of additional lineages including, cardiomyocytes, endothelial cells, hepatocytes, and neural cells. Again, the physiological relevance of these findings remains to be determined.
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Verification of Mouse Mesenchymal Stem/Stromal Cell Identity by Analysis of Mesenchymal Stem Cell Marker Expression. A. Undifferentiated mouse mesenchymal stem cells were stained with the indicated antibodies (open histograms) or the corresponding isotype controls (filled histograms) using the Mouse Mesenchymal Stromal Cell 4-Color Flow Cytometry Kit (Catalog # FMC003). The data show that mouse MSCs have the expected phenotype (positive CD105, CD29, and Sca-1; negative for CD45). B. Osteocyte-differentiated mouse MSCs were stained with the indicated antibodies (open histograms) or the corresponding isotype control (filled histograms), as described in the procedure. Osteocyte-differentiated (20 days) cells show a characteristic reduction in CD105, CD29, and Sca-1 staining. CD45 is a negative control for both cell types. |
Assay Procedure
Refer to the product datasheet for complete product details.
Reagents Provided
Reagents Supplied in the Mouse Mesenchymal Stromal Cell 4-Color Flow Cytometry Kit (Catalog # FMC003):
- Flow Cytometry Staining Buffer (100 mL)
Positive Markers
- Rat IgG2A Anti-Mouse Endoglin/CD105-Fluorescein (Clone 209701)
- Rat IgG2A Anti-Mouse Integrin beta 1/CD29-PE (Clone 265917)
- Rat IgG2A Anti-Mouse Sca-1-APC (Clone 177228)
Negative Markers
- Rat IgG2B Anti-Mouse CD45-PerCP (Clone 30-F11)
Other Supplies Required
Reagents
- Fc Receptor Blocking Reagents
Materials
- Flow Cytometry Tubes
Equipment
- Clinical Centrifuge
Procedure Overview
- Wash the cells with Flow Cytometry Staining Buffer.
Perform a cell count. - Add Fc receptor blocking reagents. If using excess pre-immune IgG to block Fc receptor, the excess IgG does not need to be washed from the cells following the incubation period.
- Transfer approximately 100 µL of the Fc receptor-blocked cells (about 1 x 106 cells) into a 5 mL flow cytometry tube.
- Add 10 µL of each fluorochrome-conjugated antibody or each corresponding isotype control antibody.
- Incubate the mixture for 30-45 minutes at room temperature in the dark.
- Wash the cells with 2 mL Flow Cytometry Staining Buffer.
- Resuspend the cell pellet in 200-400 µL of Flow Cytometry Staining Buffer.
- Analyze expression of MSC markers simultaneously by flow cytometry.
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