Hello Martha,
With regards to question 1) a pulse-label of 8 hrs is likely much
longer than necessary for a culture with a 22 hr doubling time. We pulse
cell lines with similar doubling times for 1 hr or less. As for the 72hr
(Kg1A), I would recommend trying a range of pulse-labelling times from 2-4
hrs. Your concentration of BrdU (10uM) seems appropriate. One word of
caution-- optimize and use the smallest pulse-labelling time possible.
Bromodeoxyuridine can be "disturbing" to cells in culture...
With regards to question 2), we use a protocol which uses 4N HCl with
very good results. (One technical point to note-- if your stock HCl is 12N
(for instance) it is critical to dilute the HCl immediately prior to
staining).
Lastly, an older reference which seems to be quite appropriate
(although there are many good papers on this subject) would be Tsurusawa et
al. (1988), "Flow cytometric analysis by bromodeoxyuridine/DNA assay of cell
cycle perturbation of methotrexate-treated mouse L1210 leukemia cells."
They have no trouble using PI in conjunction with BrdU to distinguish cell
cycle position.
Good luck!
Leigh Eward
Florida Institute of Technology
Cell Biology
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