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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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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web