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We have not specifically used mouse thymocytes but do have a lot of
experience with recovery of fixed human and murine cells of similar size
and/or density. After fixation, all centrifuging is performed with a large
volume of wash buffer at a minimum of 1600g for 5 min or 1000g for 8-10 min.
More critically, the centrifuge temperature is kept at 22 degrees C. We find
that lower temps (especially 4 degrees) often prevent fixed cells from
pelleting and decreases your recovery dramatically. The short spin time does
not seem to affect the cells even if other procedures require low temps.
Hope this helps!
H Rinder
Henry.Rinder@Yale.edu
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From: owner-cyto-sendout
To: Cytometry Mailing List
Subject: Help for the TUNEL method for apoptosis
Date: Tuesday, May 02, 1995 7:03PM
Recently we have been trying to setup the TUNEL method to detect
apoptosis
of mouse thymocytes in suspension, according to the published method by
Kishimoto H.
et. al. 1995 J. Exp. Med. 181:649. But we have a major problem, that is the
recovery
of cells, expecially after refixation with 1% paraformaldehyde following
initial
fixation with 70% ethanol. At this step we lose more than 95% of our
starting
cells!
So far we have tried different spinning conditions but the yield never
improved. Is
there a particular speed and/or temperature to centrifuge the cells after
the
paraformaldehyde treatment?
I would very much appreciated any suggestions.
Thanks in advance.
Derya,
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Derya Unutmaz,
Immunobiology Research Institute,
Siena, Italy.
E-mail: Unutmaz@iris02.biocine.it
Fax: +39-577-24 35 64
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