Incubate for 1 hour. Incubate for 30 mins at room temperature. Separate derivatized PE and Ab from activators on G-10 columns. You should now have approximately 2 ml of each reactant. Agitate and incubate 30 mins at room temperature 8.
Separate conjugate Apply to top of separation column.
Begin collecting 1.0 ml fractions when pink band approaches bottom of column and continue for 10 fractions after it has disappeared. Test activity of each fraction against known reactive cells by flow cytometry. Pool these fractions. Final concentration Concentrate pooled conjugate fractions using Centriprep and Centricon concentrators. Do not freeze.