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, calf, 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 permeabilized cells
   • this problem may be cell type dependent