We have experienced similar trouble in counting cells, and find
that
using a DNA stain is fine if cells are synchronized properly, else if
there
is a fair bit of polyploidy and division this proves a little
inaccurate,
on a single field
basis.
We have now resorted to some fluorescent surface stain as a suitable
marker.
On example is a fluorescenated antibosy to any known surface receptor
on
yr cells and then normalize the total fluorescence from yr field
of
interest to this value.  This method appears to work fairly
reproducibly.
Good
luck,
Jitu
Mayor                                                                  On
Mon, 13 Jan 1997, Paul White wrote:

> I was wondering if anyone could suggest a method of counting cells in a
> confocal field of view for use in an FITC-labelled-drug uptake study in
> keratinocytes. I'm finding that I need a method of counting the total number
> of cells, since some cells take up the drug very well and others don't at
> all, so its hard to estimate the proportion of cells fluorescing etc.
>
> I'm now using a two channel system so dual labelling is a possibility (eg
> with Texas Red conjugated to a live cell marker?), but I was hoping that
> there was a simpler solution, particularly since these are time course
> experiments over up to 24 hours.
>
> Thanks in advance!
>
> Paul
>
> Dr Paul White
> Research Officer
> Dermal Therapeutics R&D Programme
> Royal Children's Hospital
> Melbourne, Australia
>
>
> Loretta Donders
> Project Management Assistant
> Dermal Therapeutics R&D Programme
> The Royal Childrens Hospital
> Flemington Road
> Parkville   Vic   3052
> AUSTRALIA
> Phone:  61 3 9345 7909
> Fax: 61 3 9347 7763
> E-Mail:  [log in to unmask]
>

Dr. Satyajit Mayor                              Ph:  91 80 334-4062
National Centre for Biological Sciences                    334-5615
TIFR Centre, IISC Campus,                                  334-3138
Bangalore 560012,                                          334-2816
India                                           Fax: 91 80 334-3851
E-mail:                                   [log in to unmask]