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We are starting to flow platelets which we have never flowed and the person doing the
experiments has never prepped. In our initial experiments, we had trouble separating the
platelets from the debris. I did a lit search and it looks like most people use light
scatter to gate out the debris. This did not work for us, and I was wondering if anyone had
any idea if maybe I was doing something wrong, or maybe we just need to clean up the preps.
We are using a Coulter Elite, I tried linear light scatter and log, I tried discriminating
on FS and SS, with similar results. Based on antibody binding, we only had about 10%
platelets. Debris? Or do I need to do something to improve the FS signal? (We would
prefer not to use fluorescence to discriminate, if possible.)
Unrelatedly, we are trying to duplicate some Ca++ flux experiments that were performed on a
fluorimeter. I found a cite which says that the two techniques give similar results. Does
anyone know what the general lower limit of sensitivity is for these two methods? Or does
this have to be determined for each experiment, each cell type specifically. The
fluorimeter studies showed an immediate influx of Ca++ corresponding to less than 100nM of
Ca++. We do not see any flux on the flow.
We would greatly appreciate any input on these matters.
Thanks.
Kris Weber
University of Michigan
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