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Thanks to those that responded to my question about labeling cells
with BrdU. For those that are interested, the problem I was having
with 32D cells was due to increased resistance to the DNA denaturation
step (compared with 3T3 cells). I switched to heat denaturation (from
2N HCl, 37 C for 15 min.) on the advice of Bert Houtz at BD (thanks
Burt). This solved the problem. The conditions were 100 C for 10 min
in water (belive it or not!). Cell loss was substantial but I did get
a good BrdU signal. I may try increasing the incubation time in 2N HCl
for future experioments. Anyhow, my conclusion was that DNA
denaturation conditions may have to be varied for different cell
lines.
-Ron Hill, Ph.D.
Research Scientist
Sugen, Inc.
Redwood City, CA
ronald@sugen.sf.ca.us
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