Periosteal MSC were harvested as described from C57 Bl/6 and CD47-null mice. Post digestion, cells were resuspended in PBS and stained with a panel to mark skeletal stem cells24 (link),48 (link) using conjugated antibodies for CD90, Sca1, CD140a, CD51, CD200, Ly51, CD105, and a lineage negative cocktail. Samples were stained with fixable viability dye for 30 minutes. TrueStain FcX block (Biolegend, San Diego, CA; 156604) was added and cells were pelleted and washed prior to antibody staining at 4 °C for 1 hour. Following staining, samples were pelleted, washed, and fixed using commercially available Fixation/Permeabilization Buffer (Biolegend, 426803). Cells were analyzed on an LSRFortessa Cell Analyzer (BD), and data was analyzed using FlowJo (BD).
Protocol detail
Isolation and Characterization of Skeletal Stem Cells
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Corresponding Organization : University of Michigan–Ann Arbor
Other organizations : Michigan State University, University of California, San Francisco, City Of Hope National Medical Center
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Variable analysis
independent variables
- Mice genotype (C57 Bl/6 and CD47-null)
- Expression of skeletal stem cell markers (CD90, Sca1, CD140a, CD51, CD200, Ly51, CD105)
- Cell isolation and processing protocol (periosteal MSC harvesting, digestion, resuspension in PBS)
- Antibody staining conditions (30 minutes staining with fixable viability dye, 1 hour staining with antibodies at 4°C, blocking with TrueStain FcX)
- Cell analysis (flow cytometry on LSRFortessa Cell Analyzer, data analysis using FlowJo)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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