RE: Rhodamine123 vs. Hoechst 33342 for side population detection

Hi Albert...

 

We presented a poster at ISAC in May showing that the DyeCycle Violet
(DCV) DNA binding dye from Molecular Probes Invitrogen worked very well
for detecting SP cells using a violet laser, in both mouse and human
bone marrow and human cord blood.  The excitation peak of DCV is
slightly longer than Hoechst 33342, so you get better excitation and
clearer SP resolution.	 It gives a SP "tail" somewhat similar to that
seen with Hoechst 33342, and is restricted to LSK cells in mouse bone
marrow, as is Hoechst 33342.  I think Jonni Moore also had a poster at
ISAC that demonstrated it worked in stem cells from other species, too. 

 

We use it at 10 uM in the same buffers and incubation conditions as you
would use for Hoechst 33342.  The filters are also the same as for
Hoechst, a blue in the 450 nm range and a red in the >650 nm range.

 

I'll send you a copy of our poster if you are interested.

 

Good luck,

 

 

 

________________________________

From: Albert Tai [mailto:acktai@exelixis.com] 
Sent: Friday, October 06, 2006 12:20 PM
To: cyto-inbox
Subject: Rhodamine123 vs. Hoechst 33342 for side population detection

 

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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