KI-67 staining of HSCs

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