Hi Kimmo, I think your problems are stemming (no pun intended, honest!) from the filter set-up of the LSR. If you look at the laser paths, the UV signals are directed to the FL4/5 PMTs via a 510LP filter so only fluorescence below 510nm will get to the detectors. As you say, this is a bit of a problem when looking for signals in the far red in the SP population. The problem will be worse if you alter the filter in front of FL4 to say a 650LP; keeping a 510/20 there will give you some signal but probably not far enough out in the red for the 'classical' SP population. There is no off the shelf solution as far as I can see other than modifying the LSR considerably - I believe BD may be working on this. It would be an adavnatge to be able to do SP analysis on a benchtop and I belive that the Cytomation CyAn may be better equipped to do this - currently we do all stem cell analysis on the MoFlo. Derek On Wed, 27 Feb 2002, Porkka Kimmo wrote: > 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. ************************************************************************ Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3100 Cancer Research UK, e_mail:derek.davies@cancer.org.uk London Research Institute, mobile: 07790 604112 44 Lincolns Inn Fields, London, UK. Web Page: http://sci.cancerresearchuk.org/axp/facs/davies/index.html In tenebris lux *************************************************************************
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