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1. variaibility in antibody incubation conditions was highlighted by several
people particularly in controlling the volume left after wash steps. This
also tied in with the discussion about fluorescence linearity at
substaturating concentrations of antibody as these antibodys do not
saturate at "reasonable" antibody concentrations.
2. Variability in sample characteristics eg cell number, low vs high flow
rate sample volume were also highlighted as affecting flow
characteristics and therefore fluorescence. In our case variability in
sample volume was contributed to by leaking sheath fluid when the sheath
guard was removed.
my previous method involved acquistion in a small sample volume, at high
flow rate with considerable variaibility in cell no. Acquisition at low
rate in a larger volume with th sheath guard on seems to have resolved
the problem.
Thanks again to all who contributed suggestions
Matthew Links
Oncology Research
Prince of Wales Hospital
Sydney Australia 2031
ph 612 93822623
fax 612 93822629
email p2170227@vmsuser.acsu.unsw.edu.au
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