Sam, Most likely this is a titration (affinity) issue. The CD20 clone (L27) is very low affinity... meaning that the more you add, or the longer you wait, or the warmer temp you use for staining, the more intensity you will see. This is true on human cells: I was unable to saturate CD20 staining even by using very high concentrations of the antibody (for which you need to get highly concentrated conjugated antibody). This is not something specific to the BD clone of anti-CD20--I've seen it with multiple other clones as well. My guess is that on the NHP cells, this problem is exacerbated. I would recommend trying a staining where you use as high a concentration of antibody as possible (i.e., resuspend the cells in neat undiluted antibody, incubate for a while before adding other antibodies). In addition, trying staining at room temp or even at 37C to increase the binding rate. Finally, try staining for a much longer period of time (30-60 min) than you might otherwise. If these steps help, then you are facing the affinity problem. While I agree that the various online NHP cross-reactivity sites are very helpful, one thing that is missing is some sort of relative avidity information. It's quite possible that some antibodies labeled as non-cross-reactive actually would work at much higher concentrations; likewise, it's even possible that some antibodies could be used at significantly lower concentrations. We are in the process of titrating all of our reagents on both human and macaque to address these issues. I would encourage everyone to do the same--titrate your antibodies (on the same day, with the same concentration and incubation conditions) on both human and NHP cells. Then we can start reporting "relative avidity" and "relative saturating fluorescence intensity" information which will be highly useful to everyone doing NHP studies. This information is far more useful than the simple assertion that the antibodies do, or do not, cross-react. In particular, for example, where the cross-reactivity is seen but the avidity is much lower, researchers would know to alter incubation conditions (or to only use very bright conjugates like PE). I hope that the cross-reactivity sites (like Harvard's) will soon include this kind of information. mr
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