 |
 |
 |
 |
 |
I have been trying to phenotype lymphocytes extracted from tiny gut biopsies,
not surprisingly I am having some difficulties. I have used mechanical
disruption methods (chopping up and sucking up and down needles) with success
but as we want to do this with samples from HIV infected patients such an
approach is inadvisable to say the least. So, I am now trying to use an enzymic
method using collaginase and agitation for a couple of hours. The sample
digests quite nicely but I am getting curious results like the majority of
cells are CD19/CD3 positive! Some other markers are OK. I do my staining along
side peripheral blood from the same individual which is fine. Incidentally I
also tried using dispase to digest the tissue, worked fine but digests off CD4
and CD8. My question is does it make a difference which type of collaginase you
use and can anyone think of another reason apart from low viability for this
type of non specific staining, are there any better enzymes?. I am using four
colours, Fitc, PE, PerCP and APC.
Any suggestions? Protocols?
Simon Monard
Aaron Diamond Center
New York
|
|
|
|
|
|
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