Multi-Color Flow Cytometry Protocol to Study Mesenchymal Stem Cells within a Heterogeneous Population

R&D Flow Cytometry Kit - Flow cytometry kits designed for analysis of cell populations use up to four fluorochrome-conjugated antibodies. Flow cytometry facilitates quantitative, multiparameter analysis of cell populations based on the expression of cell surface and/or intracellular molecules. R&D Systems offers a wide range of biotin-, fluorescein-, phycoerythrin-, or allophycocyanin-conjugated monoclonal antibodies specifically designed to monitor the presence of cytokines and cytokine receptors as well as Fluorokine® kits; labeled cytokines that can be used to monitor the presence of cytokine receptors. Features of the Flow Cytometry Kit:• Simultaneous detection multiple different targets • Rapid & efficient • Single step staining R&D Fluorokine Receptor Detection Kits employ recombinant biotinylated ligands to quantitatively determine cytokine receptor density using flow cytometry. These simple kits allow the user to estimate the percentage of cells within a population which express the receptor of interest. In addition, cell surface receptor density can be assessed. Flow Cytometry Kit Contents (Multi-Color):• Fluorochrome conjugated antibodies • Isotype controls • All necessary buffers Flow Cytometry Kit Contents (Fluorokine Receptor): • Biotinylated recombinant, species specific ligand • Avidin conjugated with fluorescein (at an f:p ratio of 5:1) • Negative control reagents • Blocking antibody • Wash buffer concentrate

Product Description This kit contains four conjugated antibodies (and corresponding isotype controls) that can be used for single-step staining of human multipotent mesenchymal stromal cells (hMSCs) (1 - 4): Positive Markers • CD105-PerCP (Clone 166707; mouse IgG1) • CD146-CFS (Clone 128018; mouse IgG1) • CD90-APC (Clone Thy-1A1; mouse IgG2A) Negative Marker • CD45-PE (Clone 2D1; mouse IgG1) This kit also contains Staining Buffer (100 mL). Intended Use This product is designed for the flow cytometric analysis of hMSCs using four fluorochrome-conjugated antibodies. Storage Store at 2 - 8° C in the dark. Use within 6 months of receipt. Precaution 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. Surface Staining Protocol 1. Cell samples should be washed with 2 mL of Staining Buffer, spinning the tube at 300 x g for 5 minutes. 2. Washed cells should be counted and then Fc receptor blocking reagents may be added. If using excess pre- immune IgG to block Fc receptor, use 1 μg of IgG per 1 x 105 cells to be stained. The excess IgG does not need to be washed from the cells following the incubation period and can be carried into the staining reaction. 3. Transfer a small volume (about 100 μL) of the Fc receptor-blocked cells (about 1 x 106 cells) into a 5 mL Flow Cytometry tube. 4. Add 10 μL of each antibody or each corresponding isotype control antibody to the cells. 5. Incubate the mixture for 30 - 45 minutes at 2 - 8° C in the dark. 6. Following the incubation, remove any excess antibody by washing the cells with 2 mL of Staining Buffer. The final cell pellet is resuspended in 200 - 400 μL of Staining Buffer for flow cytometric analysis.

Product Description The StemXVivo Osteogenic supplement is a media supplement for the differentiation of human mesenchymal stem cells (MSCs) into osteocytes. All the components have been selected and optimized for human MSC osteogenesis. This product does not contain antibiotics. Intended Use StemXVivo Osteogenic Supplement is designed to be used with StemXVivo Osteogenic Base Media(R&D Systems, Catalog # CCM007) for the desired differentiation application. It may be used with other base media to differentiate MSCs depending on the experimental design of each researcher. Note: For an example using Catalog # CCM007 and CCM008 for human MSC Osteogenesis, refer to the MSC Differentiation Protocols (www.RnDSystems.com/go/OstMSCDiffProtocol). Stability and Storage Upon receipt, the StemXVivo Osteogenic Supplement should be stored at ≤ -20° C in a manual defrost freezer. The supplement can be thawed at 2 - 8° C or at room temperature. Thawed supplement can be aliquoted and stored at ≤ -20° C in a manual defrost freezer for up to 3 months. Thaw a fresh aliquot for each use. Avoid repeated freeze- thaw cycles. Limitations • FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. • The safety and efficacy of this product in diagnostic or other clinical uses has not been established. • This reagent should not be used beyond the expiration date indicated on the label. • Results may vary due to variations among human MSC progenitor cells derived from different donors.