Jose, I ran into this exact same problem myself. What I did was to try several dilutions of CFSE until I found one that gave a good labeling intensity but not so bright as to make compensation difficult or impossible. I started with 2 micromolar CFSE (in PBS) and did 2-fold serial dilutions. I found that a dilution of 0.5 micromolar worked best. Briefly, I wash the cells 2x with PBS and resuspend the cells at 20 million cells/mL in PBS (say 20 million cells in 1 mL). The I add an equal volume of 0.5 micromolar CFSE in PBS, mix and incubate 8 minutes at room temperature on a rocker. So, the final conc. of CFSE would actually work out to 0.25 micromolar. Then, I add an equal volume (e.g., 2 ml) of HI-FBS, incubate 1 minute at RT, spin, add > 10 mL of medium, spin again, resuspend in medium and then spin down an appropriate volume to stain for FACS (washing 1x with FACS staining buffer). This has worked well for me. You might want to try a similar titration yourself to see which is the highest concentration you can use and still be able to do compensation. Good luck, Larry Lawrence Allen Wolfraim, Ph.D. Laboratory of Cell Regulation & Carcinogenesis (LCRC) National Cancer Institute/National Institutes of Health 41 Library Drive Building 41, Room B1103 Bethesda MD 20892 Tel.: (301) 496-2431 Fax: (301) 496-8395 wolfrail@mail.nih.gov <mailto:wolfrail@mail.nih.gov> -----Original Message----- From: Jose Benito [SMTP:jbenito1@hotmail.com] Sent: Monday, August 26, 2002 8:50 AM To: Cytometry Mailing List Subject: CFSE analysis by flow 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? Many thanks Jose Miguel Benito Madrid Spain _____ Join the world's largest e-mail service with MSN Hotmail. Click Here <http://g.msn.com/1HM1ENXX/c157??PI=31901>
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