It's pretty much of an "all" phenomenon--the CD4 disappears, nearly
completely.
However, don't fret--we solved this problem by prestaining the cells
for the CD4 molecule. (See Mitra et al., Int Immunol 1999 11:
1801-1810 for details). Note that for this to work well, you need to
include monensin from the beginning of the stimulation: CD4 will
carry the bound antibody & fluorochrome into endosomes, which are
acidic (reducing FITC fluorescence 10 fold) and proteolytic (killing
phycobiliprotein fluorescences)--except when monensin is present,
these are both inhibited (to my knowledge, brefeldin A, which
inhibits golgi maturation, does not inhibit endosomal acidification
or proteolysis, and will not work as well. Besides, monensin is
about 1% of the cost of brefeldin). We found that CD4 cells could be
as easily distinguished after 6 hours of PMA/Ionomycin when
prestained & incubated with monensin as well as fresh unstimulated
cells.
The prestaining as well as the use of monensin from time zero did not
affect the stimulation in any way that we could detect.
It is very important to use this type of method when trying to assay
for CD4 cytokine production: relying on CD8-negative T cells is
really bad, because the CD4-CD8- T cells ("double negative", or DN)
cells produce tons of gamma-interferon and typically comprise about
10% of T cells--thus artefactually increasing the apparent CD4
production of gIFN because of incorrect identification of the CD4 T
cells!
mr
At 5:10 PM -0500 1/11/00, Maher3148@AOL.COM wrote:
>I am planning to study intracellular cytokine expression in lymphocytes
>following activation with PMA + ionomycin, but have concerns about reports
>of CD4 down regulation following activation. I would like to know if this
>is an 'all or none' phenomenon or if the CD4 remains expressed at a level
>sufficient to permit delineation from true CD4 negative cells. If you have
>experience here, I would appreciate your comments. Thanks.
>
> please reply to: kmaher@med.miami.edu
>
>
This archive was generated by hypermail 2b29 : Sat Mar 10 2001 - 19:31:03 EST