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