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- Kit select cells from hindlimbs using the HSC/HPC staining protocol.
- Stain kit selected cells with the following: CD150 PE, CD48 + Lineage FITC, Sca Percp5.5. Wash and spin 5 minutes at 1500 RPM.
- Resuspend pellet in 100 µL of cytofix/cytoperm buffer from BD. Pellets should consist of ~2 million cells.
- Wash cells by adding 1 mL 1x Perm-wash buffer from BD. Spin at 1500 RPM.
- Add Ki-67 APC antibody (1:100) and SA-PE/CY7 (1:200) to perm-wash buffer and add 100 mL per cell pellet.
- Wash by adding 1 mL of Perm-wash buffer. Spim at 1500 RPM.
- Resuspend in 0.5 mL of DAPI media (1:100, not 1:1000 in this case).
- Analyze on Aria or Fortessa.