Dear Jose, I'm very interested in your problem. From your mail I make up that you work on a Coulter machine. We did the staining in quadriple (CFSE, PE, PerCP and APC) without a problem on a BD machine. You can find an example on http://home.wanadoo.nl/flowcytometry under CFSE. It is possible to use CFSE and PE together. It might be a pain in the .... But try to get make sample to set your machine. Use bulk PBMC, label and stimulate them with SEB or PHA. Evaluate them after 4 to 5 days and you will have your settings for the next experiments. At least you will be close to them. We use 5 uM CFSE to stain them for 15 to 20 minutes. When your signal is too strong the compensation will be more difficult. Success and when you need any other suggestions.... Joost Schuitemaker __________________________________________ J.H.N.Schuitemaker Senior research technician Cellular Immunology Group Dept. Cell Biology & Histology Academic Medical Center P.O.box 22700 1100 DE Amsterdam The Netherlands Tel. + 31 20 5664960 Fax. + 31 20 ----- Original Message ----- From: Jose Benito <jbenito1@hotmail.com> Date: Monday, August 26, 2002 2:49 pm Subject: CFSE analysis by flow > > <html><div style='background-color:'><DIV>Hi flowers, I am trying > to work out a > protocol for acquisition of cells labelled with CFSE and 3 more > colors (PE, ECD and > PE-Cy5). These are PBLs stimulated with mitogens and antigens. The > problem id that CFSE > gives a staining so bright (in the fourth log decade) that > everything else (specially PE) > is completely contaminated by the strong CFSE signal. I tried last > friday to compensate > for PE channel without succes (I run out of cells to acquire). I > am going to try again > today. Does anyone have any experience working with CFSE and other > colors? Is tehre > anyway to find out a cytosettings for this combination?</DIV> > <DIV>Many thanks</DIV> > <DIV>Jose Miguel Benito</DIV> > <DIV>Madrid</DIV> > <DIV>Spain</DIV></div><br clear=all><hr>Join the world’s largest e- > mail service with > MSN Hotmail. Click Here > </html> >
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