RE: Stem cell side population detection

Hi Albert...

Our results indicate that DyeCycle Violet (DCV) works well as a SP probe
in murine bone marrow and human bone marrow and cord blood, using either
a UV or violet laser.  We haven't tried it in non-hematopoetic cells.
We use it at 10 um, but otherwise adhere to the Goodell protocol closely
(90 minutes, 37C, using her buffer).  The detection filters are also the
same - our setup is a 450/50 nm bandpass for blue, a 675/20 nm for red,
and a 580 LP dichroic.	Remember that DCV has a small but annoying 488
nm excited emission in the FITC and PE range, if you plan on doing
multiple labeling.  

DCV SP corresponds closely to the LSK phenotype in mice
(Lineage-negative, Sca-1-positive and c-kit-positive), and its efflux is
associated with the ABCG2 pump responsible for Hoechst SP.  So the
phenotypic correspondence between Hoechst SP and DCV SP seems to be
virtually identical.  These results are in press in the journal Stem
Cells.	HOWEVER, we have NOT yet done the in vivo reconstitution
experiments for DCV SP that Margaret Goodell did for Hoechst SP, to
functionally prove the existence of HSCs.

As a rule, linear scaling should be used for SP with either Hoechst or
DCV.  In log, the overall fluorescence profile is too compressed at the
high end of the scale when using reasonable PMT voltages.  Even in
linear, the appearance of the overall cell labeling is somewhat
different than that observed for Hoechst 33342, but you should be able
to recognize the SP tail.  Unfortunately my server stripped off your
attachment, but if you can resend it I can take a look.

Many groups are trying to identify stem cells in non-hematopoetic or
tumor populations using SP.  Remember, though, there are other
functional circumstances that could produce a SP-like phenotype in cells
that are not necessarily stem cells or progenitors.  As usual with flow,
try to use multiple markers for your analysis - SP alone is probably not
convincing enough.

Good luck,

Bill