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:firstname.lastname@example.org]
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:email@example.com] 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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