I'd like to (hopefully) clear up some of the confusion concerning the specific, non-non-specific and "specific-non-specific" staining associated with PE-Cy5 [or any Cy5] reagents since we have examined this in some detail at PharMingen. First with respect to the widely known "non-specific" staining observed on lymphoid tissues from virtually any species, this comes from two sources; 1) PE-Cy5 reagents tend in general to have a slightly higher "background" staining compared to other fluorochromes. This is the classic non-specific binding that can be seen with any fluorochrome-conjugated mAb and which will vary from mAb to mAb. This kind of background can be seen on any cell type, B cell, T cell, macrophage, etc. 2) PE-Cy5 (or Cy5) conjugates also have a specific affinity for Fc receptors. This results in what I refer to as specific non-specific staining. Specfic staining because it is due to a documented affinity of Cy5 for Fc receptors (At least specific in the same sense that Protein A specifically binds IgG). Non-specific staining because it has nothing to do with the specificity of the mAb being used. This binding has been directly demonstrated for human Fc receptors found on B cells and macrophages (see van Vugt, M.J. et al., 1996. "Binding of PE-Cy5 conjugates to the human high-affinity receptor for IgG (CD64)" Blood 88:2358-2360. This FcR mediated binding seems to be much worse with human PBL than with mouse tissues. That is why investigators working with human tissues are especially sensitive to this problem and why human samples are usually used to demonstrate it. While never directly proven, it is generally accepted that the high background seen in mouse tissues is also due to FcR binding because a)the background is worst on cells expressing FcR such as B cells and macrophages and b) the background can be largely blocked by mAbs to the mouse FcR. Unfortunately anti-human FcR mAbs that block this binding are not available. For reagents which show this binding to FcR the easiest way to minimize this problem (apart from using anti-FcR antibodies)is to very carefully titrate the reagent and use it at the lowest concentration possible (even below saturation if necessary). Since the FcR binding seems to have a very low affinity the background staining often titrates out much faster than the specific binding associated with the mAb. An alternative solution is to use other fluorochromes such as PerCP, PerCP-Cy5.5, PE-TR, or PE-Cy7. PerCP has no binding to FcR. PerCP-Cy5.5 and PE-Cy7 show minimal binding while PE-TR shows moderate binding (though still less than Cy5 reagents). If you have a dual-laser instrument and are only doing a three color stain, a FITC, PE, APC combination would be much better. Finally in some strains of mice there appears to be a second type of very strong specific non-specific staining of unknown origin. This artifact was first observed in NOD mice. This staining in not due to binding to FcR since it cannot be blocked by even a 100-fold excess of anti-FcR mAb and the staining pattern is not consistent with expression of FcR. Interestingly we have found the same non-specific binding in SJL and MRL/lpr mice. Notice a pattern here? We would suspect that we might see the same thing in BSXB and NZB/W mice but haven't had a chance to analyze them. I would be interested in hearing from anyone who has used PE-Cy5 reagents on either of these strains. This staining appears to be "specific" because using just pure PE-Cy5 on splenic cells the staining pattern on all of these strains is similar with minor differences. The differences seen in each strain is highly reproducible. In all strains the B cell are stained with an intensity of about 20-40X above background. In all strains all CD11b+ cells (macrophages) are negative for PE-Cy5. In all strains a small populations (2-10%) of CD3+ T cells are positive. In SJL, Gr-1 dull cells are positive while Gr-1 bright cells are negative for PE-Cy5 binding. In NOD and MRL both Gr-1+ populations are positive. In MRL mice the DX-5+ are also PE-Cy5+. What is the molecule that the PE-Cy5 is binding to? We don't know. But given its association with autoimmune prone strains of mice it might be interesting. But that's what we know up to now. Hope that this information is of some help to investigators using PE-Cy5 reagents Alan ______________________________ Alan Stall Director, Research Immunocytometry BD Biosciences-PharMingen "Mark Kukuruga" <kukuru@med.umich.edu> on 04/25/2001 03:34:54 PM To: cyto-inbox cc: (bcc: Alan Stall/SDCA/BDX) Subject: Re: PE-Cy5 in the NOD Julie, others . . . If you look at the web site, you'll see reference to Mouse, Rat, and Human reagents. Nowhere does it say that the data shown is for Human only . . . a tad misleading, no? MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Flow Core 7416 CCGC 0946 (734) 647-3216, fax (734) 936-7376 kukuru@umich.edu >>> Julie Auger <jauger@flowcity.bsd.uchicago.edu> 04/24/01 05:43PM >>> Mark - It seems this link only advertises anti-human antibodies. It has been seen by many labs, including mine, that PE-Cy5 conjugates bind to B cells from the NOD mouse strain, which Keith is referring to. Julie P.S. I also do not believe this is an Fc receptor binding, as we routinely use an antibody against Fcgamma II/III Receptor (clone 2.4G2) when we stain. At 09:23 AM 4/24/01, you wrote: >Keith, >So, are you disputing Caltag's claim? (see < www.caltag.com > ; "New Tri-Color >Conjugates" link). >MAK. > > >-- >Mark A. KuKuruga, Managing Director >University of Michigan Flow Core >7416 CCGC 0946 >(734) 647-3216, fax (734) 936-7376 >kukuru@umich.edu > > > >>> Keith Bahjat <kbahjat@ufl.edu> 04/21/01 02:59PM >>> > >Joe and others, > >As far as the binding of Cy5 to NOD B cells goes, I can tell you the new >formulations of PE-Cy5 DO NOT correct this problem . . .
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