Re: Rhodamine123 vs. Hoechst 33342 for side population detection

Attached is an illustration of some old data done with Steve Bartelmez and 
Eva Sitnicka (I presented it at a Glifca meeting years ago). Note that 
selecting the R123 low population (bottom 10% by definition) and then 
looking at the Hoechest 33342 (515nm and greater) vs. c-kit labeling of this 
R123 low populations shows a distinct population of stem cells from mouse 
bone marrow. (Note the fraction of viable, lineage-depleted cells.)

Dave
----------------------------------------
David M. Coder
Irvine, CA



>From: Ruud Hulspas <ruud-hulspas@Cytonome.com>
>To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
>Subject: Re: Rhodamine123 vs. Hoechst 33342 for side population detection
>Date: Thu, 12 Oct 2006 13:58:26 -0400
>
>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.
>
>
>
>This email (including any attachments) may contain material
>that is confidential and privileged and is for the sole use of
>the intended recipient. Any review, reliance or distribution by
>others or forwarding without express permission is strictly
>prohibited. If you are not the intended recipient, please
>contact the sender and delete all copies.
>
>
>Exelixis, Inc. reserves the right, to the extent and under
>circumstances permitted by applicable law, to retain, monitor
>and intercept e-mail messages to and from its systems.
>


This attachment - 'H33342 R123 stem cells.jpg' -  100.61 KBytes - can be viewed at
http://www.cyto.purdue.edu/MD-parts/1f453959ab4141caebcc2e52742a601d0e2a0ae6.jpg