RE: [Histonet] Cytospin - misshapen cells

From:Gayle Callis

We prefer to do a cell block and then frozen sections instead of cytospins. 
No matter how careful, gentle and little spin time, we use, the seem to 
ball up like little "BB's", and cytoplasm is very difficult to see.

We have done cell preparations where the cells relax and adhere to the 
slide surface, and that is far superior to having the cells smashed against 
the slide.  This is more time consuming but it give better cellular 
morphology.  This is with fresh, unfixed cells.

For a cell block, take 1 to 3 x 107 cells,  spin down and wash three times 
with PBS, then resuspend the last cell pellet in 1 ml OCT, immerse end of 
tube into liquid nitrogen to snap freeze. To remove the frozen cell block, 
warm end of tube slightly by holding it,  then pop the side of the tube 
against side of cryostat chamber.  The block will pop right out. IF you get 
too many cells, cut thinner sections - it can be done at 3 um.   You can 
use the wider bottom microcentrifuge tubes that hold 2 ml for a slightl 
conical but broader block face, but the method works well with other tubes 
as well.  A note:  Chris van der Loos does this method also.

Good luck

Gayle Callis HTL, HT, MT(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717

At 08:56 AM 7/30/2007, you wrote:
>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..........
>-----Original Message-----
>From: Edwards, R.E. []
>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-----
>[] On Behalf Of Martin
>Sent: 30 July 2007 14:55
>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 the file is called
>Cytospin Dapi.jpg.
>Is this normal for cytospins? Any ideas?
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