 |
 |
 |
 |
 |
I am working with several adherent cell lines including the murine
macrophage line J774A.1 and have had problems with very high background
in the FL2 parameter with the macrophages. I am using a FACS Calibur to
try and detect surface markers after trypsinizing and staining the
cells. The negative isotype control is giving me a histogram peak
channel of between 10 2 and 10 3 which is about a log scale higher than
my positive control (murine thymocytes).When I try to reduce sensitivity
by lowering FL2 voltage I lose the ability to detect the positive
control. I haven't had this high background problem in my other adherent
cell lines.
Things I have tried to reduce the background include using Fc block from
Pharmigen, blocking with rat gamma globulin, culturing the cells in RMPI
w/out phenol red, and letting the cells "heal" in complete media before
staining (I thought that membrane integrity might be comprimised after
trypsinization leading to the uptake of flourochrome conjugates).I am
using a setting of around 585 for FL2 which might be a bit hot, but any
setting much lower results in a large loss of sensitivity.
Any help or ideas would be greatly appreciated.Thanks.
Chris Reed
|
|
|
|
|
|
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