Re: Flow cytometer

["Urwin, Nigel" <nurwin@CSU.EDU.AU>]

Hi YZ
I can send you a method for leaf tissue which should work on harvested
cells next week. Most of your cells should be in G1 in both tissues.
Tissue is chopped in the presence of dye to stain DNA in released
nuclei. Relative fluoresence of the nuclei (G1 peak) is measured to
determine ploidy. Which dye you use depends on your flow cytometer and
if you are using just the one species. Preferable to use DAPI with a UV
light source or laser but propidium iodide is OK if you have the
standard 488nm laser on your FC. Trying searching the web for sites on
'ploidy analysis in plants' and you should find methods also.

Regards

Nigel Urwin
Lecturer in Plant Sciences
School of Agricultural and Veterinary Sciences
Charles Sturt University
Wagga Wagga
NSW 2678
AUSTRALIA

-----Original Message-----
From: Plant Tissue Culture [mailto:PLANT-TC@LISTS.UMN.EDU] On Behalf Of Y Shi
Sent: Friday, 7 October 2005 2:37 AM
To: PLANT-TC@LISTS.UMN.EDU
Subject:  Flow cytometer

Hi all

I want to check the chromosome number variation of canola cell suspensions.
DI Josef Schmidt suggested flow cytometer. I am going to use it. Thank you
very much, DI Josef Schmidt!

However, I do not have experience in using flow cytometry to determine cell
ploidy level. Does any one have a protocol? Any experience is very welcome.

Particularly: 1) Should I use diploid canola as standard to compare with our
suspension cells? How to normalize all the cells to G1 phase?

Thank you very much!

YZ