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Seriously, the first thing I would suggest is to do cell counts after each
step in your procedure. This can lead to revelations.
In my experience, I have found that loss occurs due to 1) centrifuging for
too long and/or too fast, 2) agitating your pellets when aspirating (this
is why I recommend decant and blot), and 3) -- very important -- excessive
washing. This last point is also important when considering the time of a procedure. I've taken it to the point where I simply increase the volume in a
tube once, spin, aand run, with no effect on the results.
Additionally, loss can be extreme when EtOH-fixing cells. Make sure you
agitate your cell pellet proir to adding EtOH; it is also helpful to keep
the cells moving while you add the EtOH.
Finally...reduce your wash volumes. Cells are often lost due to adherence
to the sides of tubes; reducing the surface area the sells come in contact
with can dramatically improve recovery.
Good luck!
Mark A. KuKuruga
Karmanos Cancer Institute
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