plant cells and probes

Iona O'Brien (iobrien@marccri.marc.cri.nz)
Fri, 15 Sep 1995 08:51:06 GMT+1200

I have been using flow cytometry to study "apoptosis" in plant
cells using a wide range of cytotoxic drugs. I am now trying to follow
physiological changes induced by these drugs (eg calcium, pH,
oxidative burst, glutathione, membrane potential) in these cells using
several probes from Molecular Probes. To complicate things I have to go
to Australia to use these probes as there is no UV laser available in Auckland.
I therefore have been trying these probes out using a fluorescent
microscope before I go.
Having played around with the technique I can observe strong
fluoresence with some of the probes, but those probes which contain
the acetoxymethyl ester (AM) eg Indo-1-AM weakly fluorescence
within the protoplasts.
Reading the literature (on plant confocal microscopy) it states that most
plants can not cleave the AM. If my controls do not fluoresce, but I can
see this weak fluorescence using these probes does that mean they are
working or am I looking at something else? The literature also states
that using pH 4.5 instead of pH 7 results in the uptake of Indo-1,
however I don't want to alter the conditions if I can help it as the
protoplasts do not like the lowered pH.
If anyone out there has any suggestions or has any experience using
physiological probes for plant flow cytometry I would welcome any
help,
Thanks in advance,
Iona O'Brien
Scientist,
Gene Transfer and Expression,
Horticulture and Food Crown Research Institute,
Auckland, New Zealand.