SP analysis

simon mccallum sm571 at hutchison-mrc.cam.ac.uk
Wed Apr 26 13:05:32 EDT 2006


Hi Devaveena,

SP resolution comes from the difference between a 'normal' hoechst channel
and a red shifted channel that picks up concentration dependent energy
transfer. The particular filters really don't matter much. Violet or blue is
fine for the normal channel and anything beyond yellow will work for hoechst
red, assuming you have a suitable dichroic to split them. I find it looks
neatest with 424/24-555DCLP-610/20, but there's not much in it. If the SP
tail exists you can't miss it. Having said that, trying a yellow or orange
filter (from your 488 emission optics perhaps) may help if the PMTs for the
UV emission lack extended range.

As to your instrument, if your LSR-II has either a violet or a 325nm HeCd
line, you might be struggling, the newer pseudo-CW light-wave UV should be
fine though. 

Of course, failing any gross problem with the specs above, your problem will
be staining related, which I'm afraid is still something of an art.

Good luck,

Simon McCallum
MRC-Hutchison Research Centre
Cambridge, UK


On 25/4/06 10:40, "Devaveena Dey" <ddey at mrdg.iisc.ernet.in> wrote:

> 
> Hi!
> We are a lab working on adult stem cells & have been trying to
> analyse the side population (SP) using Hoechst33342 staining. Though we
> ARE getting a population which seems to be disappearing in presence of
> verapamil, this Hoechst low population looks quite scattered (not the
> compact 'tail' that we see in papers). Besides, the overall scatter in the
> plot is quite high, no matter what voltage is used.
> 
> The machine we are using is the LSR2. The filter being used for collection
> of the Hoechst Red is a 675 LP, since we don't have the 690 LP which is
> normally used for collection of the 'Hoechst Red'.
> Any clue what could be the reasons for the high scatter?
> 
> DD




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