 |
 |
 |
 |
 |
Try the gentle ethanol treatment protocol described originally in Garvy et
al. (Immunology 79, 270-277, 1993) and later reviewed in Telford et al.
(J. Immunol. Meth. 172, 1-16, 1994). This method involves resuspension of
the cells in 50% FBS in PBS prior to the gradual addition of a 3-fold
amount of cold 70% EtOH. Following overnight incubation the cells can be
washed from the FBS/ethanol and resuspended in PBS (with or without a DNA
dye) for analysis. This method permeablizes the membrane and nucleus
sufficiently to allow good DNA staining while causing minimal disruption
of surface markers. We have used it extensively to simultaneously detect
cell death in mouse lymphocytes using PI or 7-AAD simultaneously with FITC
or FITC/PE immunophenotyping for surface markers. No paraformaldehyde
fixation is necessary.
Hope this helps!
Bill
On Fri, 2 May 1997, Lyndal Russell wrote:
>
> Hi everyone!!
>
> We are trying to quantitate surface marker changes simultaneously with DNA
> cell cycle analysis (using PI). We have tried this using ethanol fixation,
> but have lost the surface markers.
>
> Can anyone suggest a recipe for a better fixation method?
>
> Thanks all!
>
> Lyndal Russell
>
>
|
|
|
|
|
|
CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web