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1- Many suggested to 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. Suggestions included 150-200 g,
or 300 rpm in an Eppendorf microfuge.
2- Also a favorite is to have lots of serum around in wash buffers
used after the fixation. Sugestions 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,pipets,etc with FCS was recommended.
3- Keeping everything cold (4 degrees C, and even doing everything
in a cold room) was suggested.
4- Other points to keep in mind are -
a- since the cells are fragile , after a gentle spin,resuspend
the pellet gently.
b- a swing out bucket centrifuge is preferable to a fixed
rotor.
c- this problem can show up with detergent permiablized cells
also
d- this problem may be cell type dependant
Thanks again!
Peter Lopez
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Peter A. Lopez (617)632-3179 (voice)
Dana Farber Cancer Institute (617)632-3139 (voice)
Core Flow Cytometry Facility NO FAX!
44 Binney St. Room J-312
Boston,MA 02115 PLOPEZ@sorter.dfci.harvard.edu
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