Hi I got your e-mail from the enternit and i have a problrm with the
flowcytometry
i am trying a new drug on human breast cancer cell line 231 I incubate the
cells both the control and the drug treated cells with 10um bromodeoxy
uridine for half an hour followed by washing, trypsinization of the cell
washing twice then fixation in 70% ethanol at 2 degree centigrate then on
the day of running them in the flowcytometry usually within 2 or 3 days i
denaturate the cells with 3 n.Hcl then i add Na tetraborate then wash
twice with pbs/albumin/tween 20 then i adjust the cell number to be 1
million/1ml pbs then i add 20uL of anti Br.deoxy uridin antibody fitc
labelled and incubate for half an hour then wash twice and then adjust the
cell number to be 1 million / ml pbs then i add propidium iodide 100 uL/ 1
million cell .
the strange thing that the Pi result is very good i get a good histogram
with G1, S and G2m but for the BR.d.u nothing appear both the control
[mouse IgG] and the Fitc labelled antibody is the same very dim nothing
specific as if there is no BR.d.uridine preasent i am using coulter
flowcyt.. wave length for the bromodeoxy uridine is 525 .
the anti-br.d.ur. antibody Fitc from becton dickinson, the bromodeoxy
uridine from boeheringer.
i tried different conc of br.d.ur 20 & 30 ul i always obtain the same
results I tried to use a highier conc of Hcl but it killed the cell and
i got a lot of debris
so i hope i could find some help
my e.mail yelsh001@umabnet.ab.umd.edu
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