Mouse Mesenchymal Stem Cell Marker Antibody Panel
R&D Systems, part of Bio-Techne | Catalog # SC018
Key Product Details
Summary for Mouse Mesenchymal Stem Cell Marker Antibody Panel
For the verification of mouse mesenchymal stem/stromal cell identity by flow cytometry.
Key Benefits
- Cost-effective panel of 8 premium quality antibodies
- Defines the starting cell population
- Multiple markers reduce experimental variation
Why is it Important to Verify MSC Identity Using Established Markers?
Researchers use different techniques to isolate, culture, and differentiate mesenchymal stem/stromal cells (MSCs). Variations in experimental approaches as well as differences in the MSC starting population may account for experimental variability and some of the contradictory data that have been published in this field.
One way to minimize experimental variation is to clearly define the starting cell population by using a functional or phenotypic assay. R&D Systems offers the Mouse Multipotent Mesenchymal Stromal Cell Marker Antibody Panel, which uses expression of multiple established markers to assess MSC identity.
The Mouse Multipotent Mesenchymal Stromal Cell Marker Antibody Panel:
- Enables verification of MSC identity in less than 2 hours (with use of fluorochrome-conjugated secondary antibody).
- Includes antibodies for the positive and negative identification of MSCs.
- Uses 8 markers to increase confidence in MSC identification by flow cytometry.
- Provides a faster method to verify MSC identity compared to differentiation protocols.
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.’
Mesenchymal stem cells are multipotent, self-renewing cells that have the ability to differentiate into adipocytes, chondrocytes, and osteoblasts when cultured in vitro. Read More about MSC Nomenclature
The Mouse Multipotent Mesenchymal Stromal Cell Marker Antibody Panel includes 8 primary antibodies for the positive and negative identification of Mouse MSCs by flow cytometry.
Positive MSC Markers
- Rat Anti-Mouse Sca-1 IgG2A Monoclonal Antibody
- Rat Anti-Mouse CD29 IgG2A Monoclonal Antibody
- Rat Anti-Mouse CD44 IgG2B Monoclonal Antibody
- Rat Anti-Mouse CD73 IgG2A Monoclonal Antibody
- Rat Anti-Mouse CD105 IgG2A Monoclonal Antibody
- Rat Anti-Mouse CD106 IgG2A Monoclonal Antibody
Negative MSC Markers
- Rat Anti-Mouse CD11b IgG2B Monoclonal Antibody
- Rat Anti-Mouse CD45 IgG2B Monoclonal Antibody
Each antibody is supplied as a 50X stock; enough for 25 assays when used in 500 µL staining volume per assay. Each 1X antibody solution has an endotoxin level of
Stability and Storage
Store the unopened kit at 2 °C to 8 °C. Use within 1 year of receipt.
Opened/Reconstituted reagents may be stored for up to 1 month at 2 °C to 8 °C.
* Aliquot and store at =-20 °C in a manual defrost freezer for up to 6 months.
* Avoid repeated free-thaw cycles.
* Provided this is within 1 year from receipt.
PRECAUTION
- The acute and chronic effects of over-exposure to reagents in this kit are unknown.
- Safe laboratory procedures should be followed and protective clothing should be worn when handling kit reagents.
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.
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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Comparing and Characterizing the Phenotype of Bone Marrow Cells to Mesenchymal Stromal Cells Derived from Transgenic Mice Expressing GFP. Bone marrow cells (BMCs) isolated from C57BL/6 mice were cultured for three weeks in serum-containing mitogen-free conditions and characterized using the Mouse Multipotent Mesenchymal Stromal Cell Marker Antibody Panel (Catalog # SC018). The BMCs segregated into a population of large and small cells.A.The large cells show weak expression of Sca-1, strong expression of MSC markers CD29 and CD44, and no expression of the hematopoietic marker CD45.B.The small BMCs do not express any of the markers.C.Mesenchymal Stromal Cells from EGFP-expressing mice (MSC-EGFP cells) were derived using an established protocol1. Briefly, after BMC isolation, MSCs were isolated using frequent media changes to remove non-adherent cells. A clonal cell population was then expanded in DMEM/low Glucose containing 2% Fetal Bovine Serum (FBS), 1 nM Dexamethosone, 2.5 ng/mL Bovine FGF, 10 mg/mL EGF, and 10 ng/mL LIF. At passage 4, the MSC-EGFP cells strongly expressed CD44, and CD29, and did not express CD45. Whereas the large BMCs demonstrated weak Sca-1 expression, the MSC-EGFP cells strongly expressed Sca-1. These results suggest that despite being derived and cultured using established protocols, the MSC-EGFP cells showed a similar, but not identical phenotype to the large cells of the BMC culture. These results emphasize the importance of verifying MSC phenotypes even when using established protocols for isolating and culturing MSCs. |
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Verification of Mouse Mesenchymal Stem/Stromal Cell Identity by Analysis of MSC Marker Expression. Mouse Mesenchymal Stem Cells were stained with the primary monoclonal antibodies indicated in each panel and supplied in the Mouse Multipotent Mesenchymal Stromal Cell Marker Antibody Panel (Catalog # SC018) (filled histograms) or isotype controls (empty histograms). The cells were stained with a PE-conjugated Goat Anti-Rat Secondary Antibody (Catalog # F0105B) and analyzed by flow cytometry. The cells demonstrate the expected phenotype for mouse MSCs (positive expression of CD29, CD44, CD73, CD105, CD106, and Sca-1 and negative expression of CD11b and CD45). |
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, mouse mesenchymal stem cell marker expression can be assessed using the following procedure:
- Incubate cells with the provided antibodies for 30 minutes
- Wash the cells
- Incubate the cells with fluorochrome-conjugated secondary antibodies
- Analyze samples by flow cytometry
Reagents Supplied in the Mouse Mesenchymal Stem Cell Marker Antibody Panel (Catalog # SC018):
Positive MSC Markers
- Rat Anti-Mouse Sca-1 IgG2A Monoclonal Antibody
- Rat Anti-Mouse CD29 IgG2A Monoclonal Antibody
- Sheep Anti-Mouse/Rat CD44 Polyclonal Antibody
- Rat Anti-Mouse CD73 IgG2A Monoclonal Antibody
- Rat Anti-Mouse CD105 IgG2A Monoclonal Antibody
- Rat Anti-Mouse CD106 IgG2A Monoclonal Antibody
Negative MSC Markers
- Rat Anti-Mouse CD11b IgG2B Monoclonal Antibody
- Rat Anti-Mouse CD45 IgG2B Monoclonal Antibody
Note: These antibodies have been tested for immunocytochemistry using human induced pluripotent stem cells grown either on irradiated mouse embryonic fibroblast feeder cells (Catalog # PSC001) or in feeder-free conditions. Each antibody is supplied as a 50X stock; enough for 25 assays when used in 500 µL staining volume per assay.
Reagents
- Flow Cytometry Staining Buffer (Catalog # FC001)
- Rat IgG2A Isotype control (Catalog # MAB006)
- Rat IgG2B Isotype control (Catalog # MAB0061)
- Secondary developing reagents (Catalog # F0113, F0104B, F0105B, and F0115)
- Sterile PBS
Materials
- Flow Cytometry Tubes
- Serological pipettes
Equipment
- Benchtop centrifuge
Staining of Mouse MSC Surface Markers
Perform a cell count on harvested cells.
Resuspend harvested cells in Flow Cytometry Staining Buffer.
Aliquot 90 μL of the cells into 5 mL flow cytometry tubes.
Add 10 μL of antibody or isotype control (or a previously titrated amount).
Vortex and incubate samples for 30 minutes at room temperature.
Centrifuge samples at 300 x g for 5 minutes.
Wash the samples three times with Flow Cytometry Staining Buffer.
Resuspend each sample in 100 μL of Flow Cytometry Staining Buffer.
Add 10 μL of a fluorochrome-conjugated secondary antibody (or a previously titrated amount).
Incubate for 30 minutes at room temperature in the dark.
Centrifuge the samples at 300 x g for 5 minutes.
Wash the sample two times in 2 mL of Flow Cytometry Staining Buffer.
Resuspend the cells in 200-400 μL of Flow Cytometry Staining Buffer.
Analyze the cells by flow cytometry.
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