Re: Rhodamine123 vs. Hoechst 33342 for side population detection

Hi Albert, what two spectral regions are you planning to detect when you 
use Rhodamine123 ?
I don't think that the Rhodamine123 emission spectrum changes the same 
way as that of Hoechst33342. And that means, you won't be able to 
identify and sort cells from a 'Side Population'.
However, you can identify and sort stem cells using Rhodamine123 by 
looking for the Rho-low population in a single spectral region of it's 
emission (around 540nm). In fact, using Hoechst33342 you can do the 
same: look for the Ho-low population in a single region of it's spectrum 
(around 450nm).
Both staining procedures are very dynamic (the ratio 'dye in' vs 'dye 
out' of the cell changes over time) and thus the type of cells that fall 
within the 'low' (or 'SP') region also changes over time. It is 
therefore extremely important to design a very detailed staining 
protocol and to follow it to the letter if you want to sort a population 
that's reproducibly enriched for stem cells.
If you like, you can use both dyes in one protocol (Wolf et al., Exp 
Hematol. 1993 May;21(5):614-262)

Good luck,

Ruud

------------------
Dr. Ruud Hulspas, Ph.D.
Director of Cytometry
Cytonome
27 Drydock Ave
Boston,  MA 02210
phone: 617-330-5030 x226


From: Albert Tai <acktai@exelixis.com>
Date: Fri Oct 06 2006 - 12:20:20 EDT

Hi flow cytometer users,


I am trying to identify/sort stem cell side population (SP) using
Hoechst 33342 (based on Goodell et al.) but, unfortunately, our FACS
Aria does not equip with an UV laser.  The excitation of the Hoechst dye
by violet laser may not be optimal.  I am considering of using Rhodamine
123 for my staining and I am wondering if anyone has any success using
it for side population detection.  A point in direction for a working
protocol would be much appreciated.


Thanks you in advance for your time and assistance.


Albert

Exelixis, Inc.



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