Sonya,
This seems very complex.
Why make it too difficult. From reading your post the problem could lie
in:
1. Is the PBS isotonic
2 Freezing artefact from the (?inadequate) 10min fixation on ice.
Why not cytospin directly from the culture medium?
Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318
-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Martin
S.
Sent: Monday, 30 July 2007 11:55 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Cytospin - misshapen cells
Hi All,
I'm having some trouble with cytospins - the cells/nuclei look very
misshapen after spinning. I have used a number of different cell lines
and have the same problem with them all. I have tried fixing the cells
before spinning with 4% PFA in PBS (for this I centrifge the cells,
resuspend in PBS, centrifuge, resuspend in PFA for 10min on ice,
centrifuge, resuspend in PBS and cytospin). Unfixed cells were cytopsun
in media. The cytospin is set to 500rpm for 5min with low acceleration.
After spinning I let them air dry (~5min), wash PBS, fix 4% PFA (if not
post-fixed), wash PBS and continue with staining.
When staining the nuceli just with Dapi I get some very strange looking
nuclei, please see some pics on www.histonet.org the file is called
Cytospin Dapi.jpg.
Is this normal for cytospins? Any ideas?
Thanks
Sonya
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