Re[2]: fixing and storing cells
Susan D. DEMAGGIO (SUEDEMAG@uci.edu)
Fri, 14 Jun 96 07:53:31 PST
Or an alternative to waiting 24 hours - I just have each researcher do his
specimens at the same basic time after fixation - for example. If one
usually likes to do his immediately - then I encourage them to always do
them immediately - not wait 2 days for the next batch - in case the
characteristics of the cells DO change, and the adjustments of the machine
and the appearance of the histograms change as well. Some people you can't
convince that there is an optimum reading time for samples - they have
their own ideas. I am encouraging people to wait overnight for PI to
intercalate and "settle" down before doing cell cycle analysis - I have
seen lots of variations in cells - some permeabilize easier than others or
have more loosely coiled helixes or something. But I like the 24 hour idea
- cuts down on everyone insisting I stay until 6 to finish their samples
THAT day because that is when they stained it! I'll suggest it, Alice. Do
you get reticence to fix at all from some folks? Do you always recommend
fixation or does it depend on cell type? Your suggestions are very
helpful! THANKS
Sue
______________________________ Reply Separator _________________________________
Subject: Re: fixing and storing cells
Author: Alice.L.Givan@dartmouth.edu (Alice L. Givan) at biosmtp
Date: 6/13/96 11:13 AM
Hello Ruedi,
My general impression (but you should probably check it yourself) is that PE
and/or FITC-stained cells fixed in 1% paraformaldehyde (if kept cold and dark
after fixation) are very stable and can be read reproducibly after several
weeks or more. However, there are changes that occur rapidly upon fixation
(background fluorescence goes up a bit, PE fluorescence goes down a bit, some
cell types (eg granulocytes) change their scatter characteristics
significantly). Therefore, we always keep cells for at least 24 hours after
fixation before we analyze them -- to give time for these changes to stabilize.
Hope this helps.
Alice
Alice L. Givan
Englert Cell Analysis Laboratory
Dartmouth Medical School
Lebanon, New Hampshire
NH 03756 USA
tel 603-650-7661
fax 603-650-6130
e-mail givan@dartmouth.edu