side population wtih LSR

Van Bockstaele, Dirk Dirk.Van.Bockstaele at uza.be
Fri Mar 1 05:19:46 EST 2002


Dear Kimmo,

I haven't actually tried measuring the SP on the LSR but what I did try is
measuring red shifted hoechst fluorescence due to apoptosis.  The problem is
the same: you need UV excitation for the Hoe but you want to collect red
fluorescence, what is impossible to obtain on fl4 or fl5, because of the
wavelength specifications of the dichroics.

The trick is to collect the red fluorescence using fl3.  But in front of
this PMT's there is a "laser 1 pinhole" installed, because "normally" along
this path one collects fluorescence due to laser1 excitation .  You should
throw this out and replace it with a "laser 2 pinhole".  In this way you can
nicely collect the red fluorescence due to UV excitation.  BD has the
pinholes available.

Let me know if it also works for detecting the side population! Good luck!
Dirk

Prof. Dirk Van Bockstaele
Laboratory of Hematology
Head of Flow Cytometry / Molecular Diagnostics
Antwerp University Hospital
Wilrijkstraat 10
B-2650 Edegem
Belgium
phone 32 3 821 3900 fax 32 3 825 1148


> ----------
> Van:	Porkka Kimmo[SMTP:Kimmo.Porkka at hus.fi]
> Verzonden:	woensdag 27 februari 2002 08:06
> Aan:	Cytometry Mailing List
> Onderwerp:	side population wtih LSR
>
>
> Has anyone have experience on running a side population analysis of human
> bone marrow with the BD LSR? I'm using the Hoecst 33342 dye and a FITC
> cell
> surface marker and following the published protocol to the letter, but
> still
> cannot see any Hoechst fluorescence on the red channel. Filter setup?
> Software compensation? Any help would be much appreciated.
>
> Kimmo Porkka
> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
> Kimmo Porkka, M.D., Ph.D.
> Helsinki University Central Hospital
> Dept. of Medicine, Division of Hematology
> P.O. Box 340, 00029 HUS
> Helsinki, Finland
> kimmo.porkka at hus.fi
> +358-9-471-951029 (HUCH)
> +358-9-471-71890 (Biomedicum)
> +358-400-492-100 (mobile)
> +358-9-471-71897 (fax)
> +1-810-283-8318 (eFax)
>



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