double labeling with BrDU
kirberg at nki.nl
Thu Feb 20 13:24:40 EST 1997
In article <5ehpet$gus at newsgate.duke.edu>, dla at galactose.mc.duke.edu
(Danny Altschuler) wrote:
> Does anyone outthere have any protocol for double staining with BrDU that
> does not use the HCl denaturing step?
> Of course the BrDU staining was OK but the acid step seems to destroy
> antigenicity for my protein/antibody (BTW is an HA tagged).
> I would appreciate any suggestions.
maybe you have a look at the following paper. If memory serves me right they
do BrdU detection and surface staining together.
Tough, D. F., Sprent, J., 1994, Turnover of naive- and memory-phenotype T cells
J Exp Med 179: 1127-35
On the basis of their surface markers, T lymphocytes are divided into
subsets of "naive" and "memory cells". We have defined the
interrelationship and relative life spans of naive and memory T cells by
examining the surface markers on murine T cells incorporating
bromodeoxyuridine, a DNA precursor, given in the drinking water. Three
findings are reported. First, using a new method we show that the release
of newly formed naive T cells from the unmanipulated thymus is very low
(confirming the findings of others with surgical approaches). Second, in
thymectomized mice, T cells with a naive phenotype remain in interphase
for prolonged periods; however, some of these cells divide and retain (or
regain) their "naive" markers. Third, most T cells with a memory phenotype
divide rapidly, but others remain in interphase for many weeks.
Collectively, the data indicate that long-lived T cells have multiple
phenotypes and contain a mixture of memory cells, naive (virgin) cells,
and memory cells masquerading as naive cells.
Alternatively (that's what I normally do) just do your staining (surface
or intracellular), sort the population of interest and then do the BrdU
staining on the isolated cells. This works every time.
Joerg Kirberg Tel. 0031 - 20 - 512 19 98
The Netherlands Cancer Institute FAX 0031 - 20 - 512 20 11
Div. Molecular Genetics (H4) E_mail kirberg at nki.nl
NL - 1066 CX Amsterdam
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