BAIRDW@CLEMSON.CLEMSON.EDU ("W. VANCE BAIRD -656-4953", 803, DEPARTMENT OF HORTICULTURE, CLEMSON UNIVERSITY, CLEMSON SC 29634) (04/26/91)
We are interested in using flow-cytometry to determine DNA content in plant nuclei. We have had a difficult time releasing nuclei from the cells (comparatively small <20 u, and our physical disruption methods are inadequate) but we can make tons of single cells (yes we are now trying chemical/enzyme methods). My question is: can we believe the fluorescence readings that we acquire using whole, single cells?? We use propidium iodide and excite at 488 or 514nm. My concern is that (i) the wall affects the readings; (ii) the chloroplasts, we use leaf tissue, "autofluorese" in the red range when excited by 488 nm light (and 514?) thereby over-estimating DNA content of the nucleus; (iii) the chloroplasts/chlorophyll absorbs in the red range and therefore underestimate nuclear DNA content. Any ideas, facts, thoughts as to the whimsy or validity of using intact cells in such an effort?? Vance Baird (BAIRDW@CLEMSON.CLEMSON.EDU) Horticulture Department Clemson University Clemson, SC 29634-0375 803/656-4953; FAX 803/656-4960