RE: Calculation of C classes in endoreduplicating nuclei
kukuruga%kasle1.dnet@rocdec.roc.wayne.edu
Mon, 16 Oct 1995 13:35:03 -0400
A few pionts revisiting the multi-peak question:
1) We measured meg ploidy to 128c (7 separate peaks) using both H33342
or PI. C.V.s were better with PI, ass expected (that was using UV excitation,
by the way).
2) Let's assume (I know it's dangerous) that ONE of your plant-er's data
peaks prodominates...like, for example, the G1 peak in cells. THEN, they
use this peak as a target, placing it in a standard position. Further
assuming the staining eficiency is comparable from sample to sample,
all peaks should fall in a fairly predictable pattern, facilitating simple
marker establishment and semi-utomation of analysis.
To the UV discussion...200 to 300 mW of UV?!? Fried cells, anyone?
Seriously, I successfully ran my UV output at 30 mW, then used a neutral
density ffilter to reduce the power further to 15 mW (monitored by a
Coherent power meter) to do most of my H33342 viable cell sorting. We
were cconcerned with much with the mutagenic potential of the UV laser, and
wanted to minimize it (given also the mutagenic potential of H33342).
AND still, I saw decent 90 degree scatter in the FACS440.
3) Something I forgot to mention to the plant-people...have they tried
double staining? Using two DNA stains in cobination may improve their
resolution and reduce much of the need to background-correct.
Mark A. KuKuruga
K.C.I.
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