[Histonet] Cytospin - misshapen cells

[Martin S.]

Yes, I wondered if it was just a cytospin problem - although other
people seem to get great results with cytospins! They just don't seem to
be able to let the rest of us in on the secret!
I wanted to use the cells as a positive control so they're not that
important (I already have some positively stained cells (nice adherent
cells!), a negative control and an isotype control so........) 

For future reference whats your protocol for embedding in agarose and
when you say to treat like a tissue block do you mean a paraffin
embedded tissue block or frozen? - I have previously embedded cell
pellets in sucrose for ultrathin cryosectioning but this seems like a
lot of hassle..........

Thanks
Sonya

-----Original Message-----
From: Edwards, R.E. [mailto:ree3 <@t> leicester.ac.uk] 
Sent: 30 July 2007 15:28
To: Martin S.
Subject: RE: [Histonet] Cytospin - misshapen cells

Many  years  ago  I  tried cytospinning various  types  of  cells,
cytospinning introduces  so  many  of  its  own variables, such  as rpm,
time  of  spinning and  cell  concentration, which  markedly affected
the  outcome of  the  quality  of  the  cell  prep, and  the morphology
of  the  cell  depending on  its  position on within  the ring  that  I
abandoned  it  in  favour  of  just  smearing  the  cells
onto  coated  slides or  made  cell  pellets   embedded them  in
agarose  and  treated  it  like  a tissue  block...  

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Martin
S.
Sent: 30 July 2007 14:55
To: histonet <@t> 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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