Calman Prussin wrote: >Howard, I assume basic orange 21 stoichiometrically binds to acidic >granule proteins in the mast cell and since there are fewer granules after >degranulation, that reads out as lower fluorescence. Correct? I'm guessing that the binding is predominantly to glycosaminoglycans, but, yes, the dye should ultimately detect a decrease in the number of granules. >Another major problem with such flow assays is that if you are measuring >degranulation by drop in fluorescence of intracellular granule protein >(such as tryptase or by basic orange 21 binding) your experimental >"window" is likely to be quite small. For example, if you get >20% histamine release (say 5% release with media, 25% with activation), >that would read out as a 20% drop in mean fluorescence intensity, say from >1000 to 800 fluorescence units. Not exactly a robust response. CD63 >translocation (basotest) reads out more like a conventions degranulation >assay. A very good point. Now that I think of it, if I remember correctly, at least some aspects of degranulation (notably histamine release) are not all-or-none. When I did some experiments a few years back, eagerly anticipating that basic orange 21 would be great for degranulation assays, I was actually surprised to discover that the median fluorescence of basophil lines subjected to stimuli such as A23187 didn't change all that much from control values. My initial experience with the dye, many years earlier, was oriented toward detecting basophils and/or mast cells in mixed populations, which it does quite well. When cell samples are exposed to certain agents during preparation (e.g., ammonium chloride in lysing solutions), the granules in the basophils pretty much disappear, meaning you can't stain them with basic orange 21 or with more classical stains such as toluidine blue. I (probably naively, in retrospect), sort of assumed that degranulation in response to physiologic/pharmacologic stimuli would wipe the cells' slates similarly clean (i.e., result in a true "degranulation"), and it obviously doesn't. And, since the overall responses in which basophil and mast cell degranulation are involved are mediated at least in large part by the mediators released as a result of degranulation, the bulk histamine release measurement, particularly if correlated with cell count, is almost certainly a better indicator of biologic effects than a measurement of the amount of granule-bound dye would be on a cell-by-cell basis. >If the only tool you have is a hammer (or a flow cytometer), every problem >is a nail (or a cell waiting to be stained with mAb). Well, in the instance described immediately above, cytometry (albeit not using mAb), seems not to do the job as well as a bulk assay. But how about CD63? I gather from your statement that results with CD63 should correlate better with conventional degranulation assays (meaning histamine release), but what does that mean on a cell-by-cell basis? Is CD63 translocation all-or-none, and what would you expect the correlation between CD63 and basic orange 21 measurements to look like (the experiment is certainly feasible, especially in a system with green and red lasers)? CD63 is very popular in some circles, including among the group of investigators who have used CD63 results to support claims for the activity of homeopathic doses of activators and inhibitors, but how sturdy is the foundation on which CD63 measurements stand? -Howard
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