plant cells and probes -Reply
mohedley@spiff.pmh.toronto.on.ca
Fri, 15 Sep 1995 08:29:44 -0500
I run very similar experiments to the ones you describe, examining
oxidative stress downstream of DNA damage in human cells following
exposure to chemotherapy. The cells appear to die by apoptosis in that
they form ladders on gels.
My basic set up is to measure reative oxygen intermediates with 1?M
dihydrorhodamine 123 (which is not an esterifed probe) for 30 min, then
saturate glutathione with 40?M monobromobimane for 5 min. I display
these as dual parameter log-log displays. I use propidium iodide exclusion
to detect loss of surface membrane integrity.
In the model currently in use (ara-C treatement for 24hr) I get an initial
increase in DHR and GSH, but later the cells lose reduced GSH quite
abruptlly and then show high levels of DHR fluorescence (i.e. presumably
shift from a reducing to oxidising state). Later still, they lose DHR
fluorescence as well as GSH (i.e. presumably so metabolically wrecked
they can't make high energy electrons to reduce molecular oxygen).
Calcium measured by indo-1 only kicks up at this point.
If this scheme also applies to plant cells, then the critical events in death
by oxidative stress may be occurring upstream of calcium deregulation. In
which case you don't need to use esterified probes. Sorry I can't help
about the problem of how to measure calcium in plant cells!
Please let me know if you need more information.
David Hedley
Ontario Cancer Institute/Princess Margaret Hospital
Toronto