Re: Flow cytometry methods and refs

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

Dear all and YZ
For those that are interested below is a method I use for ploidy
analysis of lavender among other things and a couple of references for
further reading.
The Website Josef mentioned is excellent.
Also see these references
Bennett MD, Leitch IJ. 2005. Nuclear DNA amounts in angiosperms:
progress, problems and prospects. Annals of Botany 95:45-90

Dolezel J, Bartos J. 2005. Plant DNA flow cytometry and estimation of
nuclear genome size. Annals of Botany 95:99-110

Methods
1. Ploidy (cell cycle analysis) analysis of plants.

Solutions and reagents
Galbraith's buffer (Galbraith DW, Harkins KR, Maddox JM, Ayres NM,
Sharma DP, Firoozabady E. 1983. Rapid flow cytometric analysis of the
cell cycle in intact plant tissues. Science 220:1049-1051)

Contains
45mM MgCl2
30mM trisodium citrate
20mM MOPS
0.1% triton X-100
pH 7.2-7.4 with 1M KOH (not less than pH 7.0 as DNases work well at <7.0).

Propidium iodide stock (PI)
Dissolve PI (Sigma 4470) in deionised water at 1 mg/ml Store at 4oC in
dark bottle.

4',6-Diamidino-2-phenylindole (DAPI)
Dissolve DAPI at 0.1mg/ml. (5mg in 50ml of H2O, filter 0.2µm filter
and store at 20oC in aliquots)

RNase stock
Different RNases can be used.
Used Sigma 5503 RNase A at 10 mg/ml in 10 mM Tris pH 8.0. Heat 95-100oC
for 20 min to remove DNase activity. Aliquot and store at -20oC.

Dithiothreitol (DTT) stock
Dissolve DTT at 100 mM in deionised water. Stock in aliquots at -20oC.

Just prior to analysis mix the following on ice:
10ml of Galbraith's buffer
500 µl of PI
100 µl of RNase
500 µl of DTT (optional to reduce effect of oxidative effects of
releasing phenolics (browning))

*If using DAPI add 0.4ml per 10ml of Galbraith's buffer but omit PI and
RNase.

1. Chop a small piece of tissue 0.5-1cm2 in a petri-dish or weighing
boat in 1 ml of buffer. Filter through 30 um mesh or filter. Incubate on
ice for 10 mins and analyse directly.

Flow cytometer set up and quality control.
Run trout or chicken red blood cells (TRBC)as a control with the dye you
are using. CV for these should be less than 3%. These cells are
nucleated. Add 1 drop of TRBCS (can buy from Beckman Coulter) to 2ml of
Galbraiths buffer (either for PI or DAPI) filter and leave on ice for 10
min before analysis.

We have a Beckman Coulter 'Quanta 488' which is very versatile. I have
used a Partec PAI and was considering buying a Partec PAII. There are
other interesting small flow cytometers available pretty cheaply now so
shop around and test before you buy!. There is a similar Flow cytometry
listserve like this one which is worth being on if you are doing flow
regularly.
Hope this helps
Nigel

Dr. Nigel Urwin
Lecturer in Plant Sciences
School of Agriculture and Veterinary Sciences
Charles Sturt University
Wagga Wagga
NSW 2678

ph: +61-269-332450
fax:+61-269-332812
email: nurwin@csu.edu.au

-----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