double labeling with BrDU

Jorg Kirberg 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:

> Hi,
> 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.
> Thanks 

Dear Danny,
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.

jorg

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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
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