Dar Carl-Magnus: We have extensive experience with these dyes. There are no new "problems" associated with these stainings, only complications. Nearly all complications have to do with the compensation difficulties. FIrst of all, you should carefully compare Cy7PE conjugates from different manufacturers: they can be significantly different in quality and spillover requirements. Second, you should recognize that most different "lots" of Cy7PE tandems (i.e., nearly every different conjugate) will require a different compensation setting. This means you will have to collect compensation controls for each different reagent. (Note that this can be true for Cy5PE reagents as well!). You might want to design your panels so that you only use 1 or 2 different Cy7PE reagents so as to keep the different compensation settings to a minimum. Third, you will have to compensate the sample using software. You can do partial compensations on the instrument, but you will not be able to completely compensate the data. There are a couple of software products that can do the compensation. On the Mac, we use FlowJo as it keeps track of the different compensation settings necessary for each different panel, and automatically applies the correct compensation as needed. Finally, you will find that some combinations work better than others (i.e., if you are doing CD3, CD4, CD8, CD19 on the FITC, PE, Cy5PE, and Cy7PE dyes, you may find that some combinations of antibody:fluorophore work better than others). We typically put together several different combinations and evaluate them before settling on one for more extensive work. mr At 5:18 PM +0200 9/27/99, Carl-Magnus Högerkorp wrote: >Dear All, > >does anyone have any experience in analyzing preparations stained with Ab's >conjugated with PE, PE-Cy5 and PE-Cy7 at the same time. Are there any >problems associated to this kind of stainings? > > >Carl-Magnus Högerkorp >Stem cell laboratory >Department of Internal Medicine >University Hospital of Lund >Sweden
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