There is a simpler way to identify & enrich for the SP population. A novel mAb which binds to the SP stem cells is now available in PE and biotin conjugates. Please see: http://www.ebioscience.com/ebioscience/whatsnew/abcg2.html You might also find the following references helpful: Kim M, Tunquist H et al. 2002. The multidrug resistance transporter ABCG2 (breast cancer resistance protein 1) effluxes Hoechst 33342 and is overexpressed in hematopoietic stem cells. Clin Cancer Res: 8(1):22-8. Scharenber CW, Harkey MA. et al. 2002. The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hemtopoietic progenitors. Blood 15;99(2):507-12. Zhou, S., J. D. Schuetz, et al. 2001. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 7(9): 1028-34. Goodell MA, Rosenzweig M, et al. 1997. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med 3(12):1337-45 Best regards, Lily www.ebioscience.com ----- Original Message ----- From: "Derek Davies" <daviesd2@cancer.org.uk> To: cyto-inbox Sent: Thursday, February 28, 2002 2:08 AM Subject: Re: side population wtih LSR > > 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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