Flow Sorting for CSCs

Flow Sorting:

Separation/Isolation of ESA+ Human Breast Epithelial Cells

  1. Trypsinize cells (count)
  2. Spin down and wash in PBS
  3. Resuspend at 1×106 cells/ml in PBS++ (cold)

PBS++:  100ml PBS

1ml insulin

500 µl FCS

  1. Incubate cells with primary Ab 10 min at R.T.
  2. Wash by adding 4 ml PBS++ to incubating AbBCIC FACS protocol2.doc
  3. Spin and resuspend in ~300 µl minimum PBS++ (or appropriate volume).
  4. put samples on ice
  5. Bring tubes with FCS for collection.

Antibody amounts:

CD24:  20 µl/106 cells

CD44:  20 µl/106 cells

ESA: 10 µl/106 cells