The University of Iowa Carver College of Medicine

Flow Cytometry Facility

ETOH Fixation Tips

The following are suggestions compiled by Peter Lopez of the Dana Farber Cancer Institute, from an inquiry made concerning cell loss associated with 70% ETOH fixation.

  1. Use a slower spin speed, as the ETOH fixed cells are more fragile and may be ruptured by the g-forces used normally with fresh material. Use 150-200g or 300rpm in an Eppendorf microfuge.
  2. Use serum in wash buffers after fixation. Suggestions included 20%-40% serum (which could be FCS, calg, goat, horse or BSA) as well as a cushion of 50-100% FCS layered under the cells to be spun. Also precoating all tubes, pipettes, etc. with FCS was recommended.
  3. Keep everything cold (4°C, and even doing everything in a cold room).
  4. Other points to keep in mind are:
        • since the cells are fragile, after a gentle spin, resuspend the pellet gently
        • a swing out bucket centrifuge is preferable to a fixed rotor
        • this problem can show up with detergent permiabilized cells, also
        • this problem may be cell type dependent