 |
 |
 |
 |
 |
Ray:
I have had luck with in detecting PKC isoenzymes via flow
cytometry in NK cells and other lymphocytes fixed by the
following procedure:
1. Cells in microplate wells (96 V-well plate, 1-2x106 cells per well)
are washed twice by centrifugation (400 g, 5 min, 4C) in phosphate
-buffered saline (PBS), pH 7.2.
2. Resuspend cells in 200 microL PBS containing 4% (w/v)
paraformaldehyde (freshly prepared) and incubate at 37C for 15 min.
3. Wash cells in 200 microL cold (4C) 0.02% (v/v) Triton X-100 in PBS.
4. Wash next in 200 microL PBS containing 1% (v/v) Tween-20 and 1%
bovine serum albumin, pH 7.8.
5. Suspend cells in PBS containing 0.2% bovine serum albumin for
immunofluorescent staining.
I have not investigated use of gluteraldehyde.
Good luck
*************************************************************
Bruce Edwards
Scientist
The Lovelace Institutes
Albuquerque, NM 87108
Email: Bruce@lucy.tli.org
Phone: 505-262-7155
Fax: 505-262-7043
|
|
|
|
|
|
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