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We need to do intracellular calcium levels in yeast cells and it would save
a lot of time & trouble if someone could provide some tips about indo-1
loading. For instance, do you need to use protoplasts and are there any
cell membrane pumps that need to be inhibited for proper loading? We
haven't tried indo yet but I couldn't detect any fluo-3 loading on my first
attempt.
We would also like to do cell cycle measurements and would be very grateful
if anybody could provide a protocol for uniform permeabilisation of the
cells. My first attempts using freeze-thaw, at the suggestion of our
molecular biologists, were unsuccessful because of incomplete dye
penetration.
We would welcome any suggestions,
Dean R. Hewish, Cell Biologist & Flow Cytometrist.
CSIRO Biomolecular Engineering, 343 Royal Parade, Parkville, 3052,
Victoria Australia.
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