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@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@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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