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.
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..........
>From: Edwards, R.E. [mailto:email@example.com]
>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...
>[mailto:firstname.lastname@example.org] On Behalf Of Martin
>Sent: 30 July 2007 14:55
>Subject: [Histonet] Cytospin - misshapen cells
>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
>Is this normal for cytospins? Any ideas?
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