reliability
p2170227@vmsuser.acsu.unsw.EDU.AU
Thu, 05 Sep 1996 09:59:41 +1000
I would appreciate any advice regarding reducing variability of
nuclear/cytoplasmic antigen expression
I am attempting to measure expression of cyclin B/ cdc2 /p53 / p21 in the
A549 human lung cancer cell line.
Unacceptible variability in my results lead me to test the same sample on
seperate days. The problem seems to be occasional outlying results eg
triplicates 150,155,300. The CV between triplicates on the same day are
quite acceptible <10% .The machine passes its sensitivty test. The
histograms look Ok.
I have considered problems at the fixation stage (ice cold methanol 10
mins); preparation of primary,secondary antibodies or PI; or at the
acquisition stage but I can't see where such a large sporadic error that
affects all three samples on a given day creeps in.
one suggestion has been that it relates to running the sample in a small
volume 200microl , perhaps leading to vsariaibility in the flow rate.
I would appreciate any suggestions .
Thanks
Matthew Links
PhD student
Oncology Research
Prince of Wales Hospital
Sydney Australia 2031
ph 612 93822623
fax 612 93822629
email p2170227@vmsuser.acsu.unsw.edu.au
CD ROM Vol 2 was produced by staff at the Purdue University Cytometry Laboratories
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If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL,
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EMAIL robinson@flowcyt.cyto.purdue.edu
