Re: Syto dyes for plant nuclei in living cells *Vendor Response*

Posted by Rosemary.White on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Syto-dyes-for-plant-nuclei-in-living-cells-tp592149p592154.html

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Mike,

Yes, those common stains do work - sometimes.  I used to think DAPI was good for all living plant cells, but have met many plant tissues, cell cultures and protoplasts that won’t take it up.  Ditto for all the rest (including DRAQ5).  My feeling is that these dyes can enter certain types of plant plasma membranes but not others.  My guess is that the charge on these dyes keeps them out, and maybe if they were modified to appear less polar to the membrane, they’d get in more readily.

And we use propidium iodide in our live/dead cell test – it is either excluded from or takes a long time to get into living plant cells.  The plant cells it gets into quickly are dying or undergoing programmed cell death – e.g. sloughing root cap cells.

regards,
Rosemary

Rosemary White                    [hidden email]
CSIRO Plant Industry            ph.     61 (0)2-6246 5475
GPO Box 1600                       fax.     [phone]
Canberra, ACT 2601            
Australia



On 9/5/08 3:33 AM, "Ignatius, Mike" <[hidden email]> wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
For those interested I can send a recently organized listing of dyes that have seen utility for plant cell work (respond off-line).  It is fairly long and includes an extensive bibliography.  We have never made it available before, but in the queue for future publication.

For example for Nuclei:  

·         Commonly used stains for nucleic acids in plants are DAPI, Hoechst 33258, YOYO®-1 iodide, YO-PRO®-1 iodide , acridine orange, ethidium bromide, and propidium iodide.  

But as Christian points out, these may not work.  However a new reagent has emerged that might be of interest.   It isn't a simple stain, but it readily labels dividing plant cell nuclei (alfalfa) in a workflow similar to BrDu but much improved with our new Click-iT technology.

Full text available at:  http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf/bioprobes/bioprobes55.Par.79287.File.dat/bp55_1_EdU.pdf <http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf/bioprobes/bioprobes55.Par.79287.File.dat/bp55_1_EdU.pdf>  
From that article:  Figure 5—Cell wall digestion is not required with Click-iT™ EdU. Medicago sativa (alfalfa) suspension cultures were incubated with 10 μM EdU for 3 hours. Cells were then fixed and permeabilized. EdU that had been incorporated into newly synthesized DNA was detected with the Click-iT™ EdU Alexa Fluor® 488 Imaging Kit (green fluorescence, Cat. no. C10083). Nuclei were stained with blue-fluorescent DAPI. Six confocal sections were overlaid onto a differential interference contrast image. Image contributed by Ferhan Ayaydin, Cellular Imaging Laboratory, Biological Research Center, Szeged, Hungary.

Kind Regards,

Mike Ignatius, Molecular Probes/Invitrogen

Eugene, Oregon, Site of the 2008 US Track and Field Trials.

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal <http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal>  I'll echo Tobias here.  It's very tough to get any dye into living plant cells and keep them that way.  We've tried nearly every nuclear stain available along with various other markers and trackers, such as mitotracker, all with no luck in the hands of the users.

Have you thought about making stables with RFP in the nuclei?  Three channel sequencing then allows you to visualize GFP, RFP and cholorplasts in the far red channel.  

Christian