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Hi,

I'm working with an investigator to determine percent viability of
chondrocytes in articular cartilage plugs.  We are trying this both by flow
cytometry and fluorescence microscopy.

For FACS analysis we incubate cartilage overnight with collagenase and then
stain dissociated cells with Molecular Probes Live/Dead reagents (calcein-AM
and ethidium homodimer-1).

However, the investigator would also like to have a 'number' for the
viability of chondrocytes in cartilage plugs (rather, slices from these
plugs) using the same live/dead reagents and looked at by confocal
microscopy.

Attached are a couple of examples.  The first (Cart5) represents analysis of
a plug from a human sample (we don't have FACS results from that day).

The second is a plug from a pig (used to try and get the technique
optimized).  Cells released from another pig sample taken the same day (same
pig) indicated 86% viability by FACS.  So the pig works well (if we were
studying healthy pigs we wouldn't have a problem!).  FACS on the pig is
shown in the third attachment.

However, with the human samples it looks as if it's going to be virtually
impossible at this time to get staining to the point where we will have an
image consisting of live and dead cells sufficiently resolved to apply a
software program to the analysis resulting in numbers of live/dead.

Nevertheless, if we do get to the point where the staining of human samples
gives more clear cut resolution, can anyone offer suggestions about
software - even if it's simply to say the % red area vs % green area).

And along the same line, another (microbiology) investigator is interested
in software with similar potential for 'counting' red and green Salmonella
(stained with live/dead reagents) in a 'smear' on a slide (please see fourth
attachment).  Any thoughts on this would be appreciated as well.

Thanks for any suggestions/advice.

Ray Hester
Univ. of South Alabama
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