Hi folks- I have been tearing my hair out with a question regarding isotype gating, so I thought, at the risk of demonstrating my inexperience in these matters, I would throw it out there for those of you with more experience in cytometry. Isotype control fluorescence subtraction has been mandated, at least in our case, by the FDA. I think I have a pretty good idea about how the group feels about isotype control usage, so we don't need to rehash that. Suffice to say that we are matching isotype and 1:1 protein/fluorochrome ratios against each antibody of interest in assays for quantitation. The question is, if your antibody of interest targets an activation marker, with your population running from "dim" to "bright" staining cells, what dictates position for region placement? Rationally, I would consider anything above isotype to be positive by definition, but what to do about that potentially fairly dense portion of the "dim" cells which overlap the area where Fc binding is prevalent? I.e. is it better to cut into the isotype negative population or the "dim" staining cells? The actual subtraction takes place once the Geo Means are converted to Number of Molecules via the bead-derived calibration curve. I have included some plots in Word format. Thanks for your indulgence! Ann Fairchild Sr QC Analyst Dendreon Corporation
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